INTRODUCTION
In the Mediterranean Sea the genus Patella comprises four species: Patella rustica (Linnaeus, 1758); P. caerulea (Linnaeus, 1758); P. ulyssiponensis (Gmelin, 1791); and P. ferruginea (Gmelin, 1971) (Sella et al., Reference Sella, Robotti and Biglione1993; Ridgway et al., Reference Ridgway, Reid, Taylor, Branch and Hodgson1998). The latter, with its present geographical distribution limited to the western basin, is considered to be at serious risk of extinction (Templado et al., Reference Templado, Calvo, Garvía, Luque, Maldonado and Moro2004). The other three congeners co-occur on rocky shores but have different vertical zonation; P. rustica occurs in the upper intertidal, P. caerulea inhabits the lower mid-littoral as well as upper infralittoral, while P. ulyssiponensis inhabits the low intertidal shore and infralittoral fringe (Della Santina et al., Reference Della Santina, Sonni, Sartoni and Chelazzi1993; Šimunović, Reference Šimunović1995; Mauro et al., Reference Mauro, Arculeo and Parrinello2003).
Patella rustica ranges from the Mediterranean to the Atlantic coasts of the Iberian Peninsula and northern Africa, including the Macaronesian Islands (Ridgway et al., Reference Ridgway, Reid, Taylor, Branch and Hodgson1998; Lima et al., Reference Lima, Queiroz, Ribeiro, Hawkins and Santos2006), but recent distributional shifts related to climate change have been recorded in Portugal (Lima et al., Reference Lima, Queiroz, Ribeiro, Hawkins and Santos2006; Sousa et al., Reference Sousa, Seabra, Wethey, Xavier, Queiroz, Zenboudji and Lima2012). Intertidal communities are fundamentally structured by limpet grazing (Hawkins et al., Reference Hawkins, Hartnoll, Kain, Norton, John, Hawkins and Price1992; Paine, Reference Paine2002; Jenkins et al., Reference Jenkins, Coleman, Burrows, Hartnoll and Hawkins2005; Coleman et al., Reference Coleman, Underwood, Benedetti-Cecchi, Åberg, Arensa, Arrontes, Castro, Hartnoll, Jenkins, Paula, Della Santina and Hawkins2006), hence they are rightfully considered to be a keystone species (sensu Power et al., Reference Power, Tilman, Estes, Menge, Bond, Mills, Gretchen, Castilla, Lubchenco and Paine1996), whose removal or alterations of behaviour may have fundamental ecosystem consequences.
A number of studies have been conducted in order to investigate the reproductive cycles of Patella congeners, e.g. Patella vulgata (Orton et al., Reference Orton, Southward and Dodd1956; Thompson, Reference Thompson1980; Bowman & Lewis, Reference Bowman and Lewis1986; Delany et al., Reference Delany, McGrath, O'Riordan, Myers and Myers2002; McCarthy et al., Reference McCarthy, Woosnam and Culloty2008; Ribeiro et al., Reference Ribeiro, Xavier, Santos and Hawkins2009), Patella depressa (Orton & Southward, Reference Orton and Southward1961; Bowman & Lewis, Reference Bowman and Lewis1986; Brazão et al., Reference Brazão, Boaventura, Morais, Narciso and Ré2003; Moore et al., Reference Moore, Thompson and Hawkins2007; Ribeiro et al., Reference Ribeiro, Xavier, Santos and Hawkins2009), Patella ulyssiponensis (Evans, Reference Evans1953; Thompson, Reference Thompson1979; Bowman & Lewis, Reference Bowman and Lewis1986; Guerra & Gaudêncio, Reference Guerra and Gaudêncio1986; McCarthy et al., Reference McCarthy, Woosnam and Culloty2008; Ribeiro et al., Reference Ribeiro, Xavier, Santos and Hawkins2009), Patella caerulea (Frenkiel, Reference Frenkiel1975; Belkhodja et al., Reference Belkhodja, Jaafoura, Missaoui and Romdhane2011) and Patella ferruginea (Espinosa et al., Reference Espinosa, Guerra-García, Fa and García-Gómez2006; Guallart et al., Reference Guallart, Calvo, Acevedo and Templado2013). The reproductive cycle of P. rustica has been previously described on the Algerian coast of the Mediterranean Sea (Frenkiel, Reference Frenkiel1975), and on the Basque (Othaitz, Reference Othaitz1994) and Portuguese coasts of the north-east Atlantic (Ribeiro et al., Reference Ribeiro, Xavier, Santos and Hawkins2009), based only on macroscopic examination of gonads and the staging scheme developed by Orton et al. (Reference Orton, Southward and Dodd1956). Although macroscopic examination based on the size of gonads allows for the description of gonad development, histology provides a more precise and accurate evaluation of gonad developmental stages.
To the authors' best knowledge, no previous research has been performed to describe reproductive cycle of P. rustica based on histological analysis, and none of the studies were conducted on limpets from the Adriatic shore. The aim of this study, therefore, was to investigate the reproductive cycle and to develop a staging scheme for gonad development of P. rustica, combining qualitative and quantitative histological techniques. Sex-ratio, gonad developmental stages, mean gonad index, oocyte size and shell length at sexual maturity were analysed in order to provide new information on the reproductive biology of this species.
MATERIALS AND METHODS
Study site and sampling
The limpet Patella rustica was hand collected from the high shores of Zaton Bay in the south-eastern Adriatic (42°41′40″N 18°02′32″E). Sampling was performed monthly, from July 2011 to June 2012. Seawater and air temperatures were obtained from the Croatian Meteorological and Hydrological Service. Additionally, chlorophyll-a concentration (chl a) at the seawater surface was determined from 500 ml sub-samples filtered through Whatman GF/F glass-fibre filters and stored at −20°C. These were homogenized and extracted in 90% acetone for 24 h at room temperature, analysed fluorometrically with a Turner TD-700 Laboratory Fluorometer (Sunnyvale, CA) calibrated with pure chl a (Sigma) and calculated according to the method of Jeffrey & Welschmeyer (Reference Jeffrey, Welschmeyer, Jeffrey, Mantoura and Wright1997).
A total of 480 specimens were sampled for histological analysis: (a) 30 individuals were sampled monthly (N = 355, mean length ± standard deviation (SD) = 24.2 ± 2.9 mm) except in December 2011, when only 25 individuals were collected due to severe weather conditions; (b) smaller-sized limpets within the range of 10.1–22.4 mm in shell length (N = 95, mean length ± SD = 16.2 ± 3.0 mm) were sampled in September–November 2011 and February, March and May 2012 and used for qualitative histological analysis. Out of these 95 smaller-sized limpets, 65 specimens collected in September–November 2011 were used for estimating the smallest size at sexual maturity and for comparison of the sex-ratio with the shell length–frequency distribution of males and females.
Histological analysis
Limpets were dissected and gonad tissue was fixed in 10% buffered formalin. Fixation was followed by standard tissue processing that included dehydration in increasingly concentrated ethanol, clearing with chloroform, embedding in paraffin, sectioning at 5 µm and finally staining using haematoxylin and the eosin method. Sections were examined at 50× 100× and 400× magnifications, sexed and assigned to a developmental stage adopted from McCarthy et al. (Reference McCarthy, Woosnam and Culloty2008) and Belkhodja et al. (Reference Belkhodja, Jaafoura, Missaoui and Romdhane2011) and modified for this species (Table 1). Five numerical stages were determined for males (Figure 1): early active (3), late active (4), ripe (5), spawning (2) and spent (1), and seven for females (Figure 2): inactive (0), early active (3), late active (4), ripe (5), atresic (1.5), spawning (2), and spent (1). Individuals whose sex was impossible to determine were classified as undifferentiated. An undifferentiated limpet could be an adult in the resting stage of gonad development or a juvenile that had not yet reached sexual maturity (see also McCarthy et al., Reference McCarthy, Woosnam and Culloty2008). A mean gonad index (MGI) was calculated monthly for males and females to estimate the proportion of individuals in different reproductive stages. Values of MGI were obtained by multiplying the number of individuals at each developmental stage by the numerical ranking of that stage, and dividing the result by the number of individuals of each sex (Gosling, Reference Gosling2003). For quantitative analysis, the size–frequency distribution of maximum oocyte diameter was studied. Oocytes with visible nuclei within one random visual field at 100× magnification (0.585 mm2) were measured using Axio Vision Rel 4.8 software in all P. rustica females sampled.
Photomicrographs of gonad developmental stages in male Patella rustica: (A) early active; (B) late active; (C) ripe; (D) spawning; (E) spent. SPG, spermatogonia; TW, tubular wall; TL, tubular lumen; SPG, spermatogonia; SPC, spermatocytes; SPD, spermatids; SPZ, spermatozoa; SPZ T, spermatozoa tails; CT, connective tissue; H, haemocytes; RS, residual spermatozoa. Scale bar: 50 µm.
Photomicrographs of gonad developmental stages in female Patella rustica: (A) early active; (B) late active; (C) ripe; (D) atresic; (E) spawning; (F) spent; (G) inactive. AW, acinus wall; OG, oogonia; CT, connective tissue; PO, previtellogenic oocyte; VO, vitellogenic oocyte; AO, atresic oocyte; ES, empty space; DO, degenerative oocyte. Scale bar: 100 µm.
Histological appearance of gonad developmental stages in Patella rustica (adopted and modified from McCarthy et al., Reference McCarthy, Woosnam and Culloty2008 and Belkhodja et al., Reference Belkhodja, Jaafoura, Missaoui and Romdhane2011).
Data analysis
Sex-ratios were tested using a chi-square (χ2) goodness of fit test. To estimate the smallest size at sexual maturity, data were fitted to the equation
$$P = 1/\lpar 1+e^{\lpar a - b \times L\rpar }\rpar \comma \;$$where P is the probability that individuals were sexually mature and L is their length. Estimated length at which 50% of analysed individuals were mature was calculated as L 50% = a/b (Sparre & Venema, Reference Sparre and Venema1998). Spearman's correlation analysis was applied to the dataset to determine the degree of association between MGI and hydrographic parameters, and between female MGI and oocyte size. In all statistical analysis, critical probability value was set at 0.05.
RESULTS
From a total of 355 specimens collected monthly, 39 individuals (10.9%) were males, 170 individuals (47.9%) were females, three individuals (0.8%) were identified as hermaphrodites, 142 individuals (40.0%) were classified as undifferentiated while one individual (0.3%) was lost in processing (Figure 3). Sex-ratios were calculated only for the specimens with maximum gonadal activity sampled from September to November to avoid sex-biased conclusions. The χ2 test showed statistically significant differences (χ2 = 7.86, P = 0.005) in the proportion of females (N = 56) and males (N = 30) with a female:male ratio of 1.9:1. In addition, gonad samples were collected from the 95 smaller-sized individuals, out of which 40 were males (42.1%), 9 were females (9.5%) and 46 were classified as undifferentiated (48.4%). The χ2 test calculated for the specimens with maximal gonad activity showed statistically significant differences (χ2 = 23.17, P < 0.001) between the number of females (N = 7) and males (N = 40) with a female:male ratio of 1:5.7. The estimated shell length at which 50% of analysed males were sexually mature was 13.1 mm. Comparing the sex-ratio and the shell length–frequency distribution of males and females, it can be seen that males dominate the smaller-sized class while females become prevalent as size increases, from ~28 mm onwards (Figure 4).
Frequency of Patella rustica females, males, undifferentiated individuals and hermaphrodites, according to sampling month (mean shell length ± standard deviation = 24.2 ± 2.9 mm).
Female:male ratio and frequency distribution of male (M) and female (F) Patella rustica with respect to shell length.
Histological analysis showed that P. rustica has one reproductive cycle per year. The frequency distribution of different gametogenic stages observed in females and males throughout the study period is illustrated in Figure 5. Female gametogenesis started in February and by May 96% of females were in early developmental stage. From February to May no males were recorded, while all males sampled in June and August had gonads in early developmental stage. Females went through ripening from September, when 70% of sampled females were in late developmental stage, to October when 67% of females had ripe gonads. During ripening, 5% of female gonads were in the atresic stage, characterized by a majority of previtellogenic oocytes and mature oocytes undergoing atresia or even complete citolysis. All males sampled in September were in the late developmental stage, and in November 60% of males had ripe gonads. The spawning peak occurred between November and December for both females and males. In December and January, all individuals had either spent gonads or were in the inactive stage.
Monthly variation in the frequency of different gametogenic stages and mean gonad index in Patella rustica (mean shell length ± standard deviation = 24.2 ± 2.9 mm): (A) males; (B) females.
The frequency distribution of oocyte diameter according to sampling month is presented in Figure 6. Monthly mean oocyte diameter ranged from 8.1 µm (January) to 118.7 µm (October). Oocytes increased in size from September (63.8 ± 40.1 µm) to November (104.5 ± 49.6 µm). After April, the frequency distribution of oocyte size had a relatively uniform pattern until October, illustrating a continuous process of maturation and release of gametes in November. There was a significant positive correlation between oocyte diameter and female MGI (Spearman r = 0.685, P = 0.014).
Monthly variation in the frequency of oocyte diameter in Patella rustica. N, number of oocytes; X, mean oocyte diameter ± standard deviation.
Analysis of environmental parameters at the sampling site (Figure 7) showed that the mean seawater surface temperature ranged from 12.5°C in February to 25.8°C in September, while the mean daily air temperatures ranged from 7.5°C in February 2012 to 27.1°C in August 2011. Chl a values ranged from 0.03 µg l−1 in September to 1.68 µg l−1 in April.
Monthly variation of mean daily surface seawater temperature, air temperature and chlorophyll-a (chl a) concentration values from July 2011 to June 2012.
DISCUSSION
The present study investigated the annual reproductive cycle of Patella rustica using standard qualitative and quantitative histological techniques as the primary method for staging gonad development. The same method has been applied to several other limpet species, such as Patella vulgata, Patella ulyssiponensis (McCarthy et al., Reference McCarthy, Woosnam and Culloty2008) and Patella caerulea (Belkhodja et al., Reference Belkhodja, Jaafoura, Missaoui and Romdhane2011), but not to P. rustica. Histology allows a more precise evaluation of gonad developmental stages, especially early developmental stages, that might be overlooked by other methods (McCarthy et al., Reference McCarthy, Woosnam and Culloty2008), such as macroscopic examination of gonads according to the scale established by Orton et al. (Reference Orton, Southward and Dodd1956).
Results showed that P. rustica gonads have only one reproductive cycle per year. The reproductive cycle includes a longer period of gamete development from February to October for females and from June to November for males, with a spawning peak in November/December for both sexes and gonad redevelopment from January. During gonad redevelopment, most limpets had undifferentiated gonads, probably in the post-spawning and inactive or resting periods. While for females it is possible to histologically detect inactive gonadal stage due to the presence of oogonia, this is not the case for male gonads where only connective tissues and undifferentiated gametes are present. Consequently, this would explain the lack of males in the samples from February to May and in July (see Figure 5). Undifferentiated limpets, with a higher percentage in the post-spawning period than with gonads maturing, have been reported in previous studies on P. vulgata (53% of specimens in the post-spawning period, McCarthy et al., Reference McCarthy, Woosnam and Culloty2008), P. ulyssiponensis (80% of specimens in the post-spawning and early development periods, McCarthy et al., Reference McCarthy, Woosnam and Culloty2008) and P. caerulea (51% of specimens in the spawning and post-spawning periods, Belkhodja et al., Reference Belkhodja, Jaafoura, Missaoui and Romdhane2011). In addition, the gap between early development of females and males could be due to the fact that female gonads usually develop more slowly than male gonads of similar size (Bowman, Reference Bowman, Moore and Seed1985; McCarthy et al., Reference McCarthy, Woosnam and Culloty2008; Belkhodja et al., Reference Belkhodja, Jaafoura, Missaoui and Romdhane2011).
Findings from this study confirm the general reproductive pattern of P. rustica described previously on Algerian (Frenkiel, Reference Frenkiel1975), Basque (Othaitz, Reference Othaitz1994) and Portuguese coasts (Ribeiro et al., Reference Ribeiro, Xavier, Santos and Hawkins2009), but also showed a few marked differences. This study suggests that P. rustica in the Adriatic Sea has a longer gametogenic period, suggested also by Frenkiel (Reference Frenkiel1975) and Othaitz (Reference Othaitz1994), as opposed to the shorter period reported by Ribeiro et al. (Reference Ribeiro, Xavier, Santos and Hawkins2009). The longer gametogenic period could result from no inhibitory effect of extreme low temperatures on gonad development, as suggested by Ribeiro et al. (Reference Ribeiro, Xavier, Santos and Hawkins2009) for P. depressa and P. ulyssiponensis, since the mean seawater surface winter temperature (December–March) in the Adriatic Sea was 14.9°C. There was, however, no evidence of multiple spawning events between August and November, as reported by Othaitz (Reference Othaitz1994). The seasonality of spawning varies between species and with seawater temperature, causing the timing of spawning to vary depending on latitude (Guerra & Gaudêncio, Reference Guerra and Gaudêncio1986).
Temperature has often been linked to the reproductive cycles of Patella species (Orton et al., Reference Orton, Southward and Dodd1956; Fretter & Graham, Reference Fretter and Graham1976). Thus, P. rustica has only one spawning peak near its northern range (Ribeiro et al., Reference Ribeiro, Xavier, Santos and Hawkins2009) and in the present study, as opposed to multiple spawning events on the warmer Mediterranean coasts (Frenkiel, Reference Frenkiel1975; Othaitz, Reference Othaitz1994). Spawning is often associated with strong wave action (Orton et al., Reference Orton, Southward and Dodd1956; Orton & Southward, Reference Orton and Southward1961; Branch, Reference Branch1974) and a drop in seawater temperature (Bowman, Reference Bowman, Moore and Seed1985; Delany et al., Reference Delany, McGrath, O'Riordan, Myers and Myers2002). Sea roughness as a trigger for spawning can be extremely important for high shore species (Branch, Reference Branch1981; Shanks, Reference Shanks1998), such as P. rustica. Maturation and spawning can be correlated with food abundance: maturation when there is enough food supply for the necessary higher energy input, while spawning just prior to the phytoplankton bloom ensures food for planktotrophic larvae (Branch, Reference Branch1974, Reference Branch1981). However, site-specific variations are likely to contribute to the rate of gonad development, as well as the timing of spawning (McCarthy et al., Reference McCarthy, Woosnam and Culloty2008). Findings from this study showed no concurrence between the reproductive cycle of P. rustica and environmental parameters, although the reproductive patterns generally fit the latitudinal trend previously described for Patella species, where spawning occurs later in the year, possibly triggered by low temperatures and/or sea roughness.
A number of limpet species are thought to be hermaphrodites, and most are considered to be protandrous, including P. caerulea (Bacci, Reference Bacci1947; Belkhodja et al., Reference Belkhodja, Jaafoura, Missaoui and Romdhane2011), P. ulyssiponensis (Thompson, Reference Thompson1979; McCarthy et al., Reference McCarthy, Woosnam and Culloty2008), Helcion pectunculus (Gray & Hodgson, Reference Gray and Hodgson2003), Lotia gigantea (Kido & Murray, Reference Kido and Murray2003) and Patella ferruginea (Rivera-Ingraham et al., Reference Rivera-Ingraham, Espinosa and García-Gómez2011a). The sex-ratio and shell length–frequency distribution of male and female P. rustica showed that males dominated the smaller-sized class while females became prevalent with increasing size, from ~28 mm onwards. The preponderance of larger females could suggest that P. rustica is a protandrous hermaphrodite, although assumptions based only on size–frequency distribution provide no direct evidence of protandry (Le Quesne & Hawkins, Reference Le Quesne and Hawkins2006). The hermaphroditic phenomenon is most likely regulated by other factors, such as environmentally and genetically determined mechanisms (Lindberg & Wright, Reference Lindberg and Wright1985; Rivera-Ingraham et al., Reference Rivera-Ingraham, Espinosa and García-Gómez2011a). A male-biased sex-ratio, frequent for protandric patellids, was observed only in smaller-sized P. rustica. Estimated length at which 50% of males were mature was 13.1 mm, and according to the age–length key for this species these males were younger than 2 years (Prusina, Reference Prusina2013). Similar findings were demonstrated for other protandrous limpet species (Blackmore, Reference Blackmore1969; Gray & Hodgson, Reference Gray and Hodgson2003). However, a considerable percentage of males found among the larger-sized classes indicate the possibility that some specimens never change sex (Espinosa et al., Reference Espinosa, Rivera-Ingraham, Fa and García-Gómez2009) or are capable of two-way sex change (Le Quesne & Hawkins, Reference Le Quesne and Hawkins2006), a common phenomenon found in P. ferruginea (Guallart et al., Reference Guallart, Calvo, Acevedo and Templado2013). This reverse sequential hermaphroditism could help regulate the population dynamics by controlling the sex-ratio and maximizing the reproductive fitness under different environmental conditions (Guallart et al., Reference Guallart, Calvo, Acevedo and Templado2013). What causes change of sex and what is the adaptive significance of protandry is still not completely explained. Generally, protandry is considered to be beneficial, especially to broadcast spawners and species relying on an r-strategy, such as P. rustica, since larger females would be able to produce a greater number of energetically more expensive eggs (Hoagland, Reference Hoagland1978).
Since qualitative histological methods of determining the reproductive stages may be to some extent subjective (Gosling, Reference Gosling2003), a quantitative method of measuring the size of oocytes (expressed as oocyte diameter) was also employed. In this study, quantitative analysis followed the pattern observed using qualitative histological methods. Part of the oocytes undergo lysis and different stages of atresia during the whole period of development, and particularly at the beginning of the spawning season. Oocytary atresia is not unusual and has been reported for other limpet species (see Morriconi, Reference Morriconi1999; Belkhodja et al., Reference Belkhodja, Jaafoura, Missaoui and Romdhane2011). Although it is known that oocyte size can be influenced by life strategy, location or environmental factors, comparisons can be made only with other limpet species, since this is the first assessment of oocyte size for P. rustica. Similar oocyte size and bimodal pattern of oocyte distribution was reported for other limpet species, such as Patella granularis or H. pectunculus (Vat, Reference Vat2000; Gray & Hodgson, Reference Gray and Hodgson2003). Many limpet species are known to have high fecundity, with oocyte diameter <180 µm, suggesting these species produce planktotrophic larvae (Branch, Reference Branch1974). Spawning in such species is considered to be correlated and/or triggered with the peak of primary production (Brêthes et al., Reference Brêthes, Ferreyra and de la Vega1994; Morriconi, Reference Morriconi1999), although some studies suggested that veligers are non-feeding, so that spawning and the phytoplankton bloom are not necessarily correlated (Hadfield et al., Reference Hadfield, Strathmann and Strathmann1997). The latter may explain the absence of correlation between spawning of P. rustica and chl a concentration in the study area.
Coastal natural resources are under unsustainable pressures nowadays, the harvesting of intertidal macroinvertebrate resources being one of them (Katsanevakis et al., Reference Katsanevakis, Lefkaditou, Galinou-Mitsoudi, Koutsoubas and Zenetos2008). Uncontrolled harvesting and poor management measures have led to the destructive decline of several limpet species, such as Patella candei candei in the Canary Islands (Hawkins et al., Reference Hawkins, Corte-Real, Pannacciulli, Weber and Bishop2000; Navarro et al., Reference Navarro, Ramirez, Tuya, Fernandez-Gil, Sanchez-Jerez and Haroun2004), P. ulyssiponensis in the Azores, and even to the risk of extinction, such as P. ferruginea, whose distribution has been extremely reduced during the 20th Century (Templado et al., Reference Templado, Calvo, Garvía, Luque, Maldonado and Moro2004; Espinosa & Ozawa, Reference Espinosa and Ozawa2006; Rivera-Ingraham et al., Reference Rivera-Ingraham, Espinosa and García-Gómez2011b). One of the reasons for their increased vulnerability is their protandric life history, since larger individuals have greater probability of being collected, resulting in recruitment failure due to decreased reproductive output (Hawkins et al., Reference Hawkins, Corte-Real, Pannacciulli, Weber and Bishop2000). Although limpets in Croatia are currently not commercially exploited, they are either systematically or occasionally collected for human consumption or as fishing bait (Zavodnik & Kovačić, Reference Zavodnik and Kovačić2000).
The present study is vital for the rational management of stocks, since P. rustica is considered to be a keystone species on Mediterranean rocky shores, with potential for commercial exploitation. Additionally, it is the first histological description of gonad developmental stages and the first description of oocyte size and distribution in P. rustica. Data gathered give a more detailed insight into the reproductive biology of P. rustica and contribute to our understanding of the biology of Patella species. Reported findings can serve as a good baseline and point of comparison for studying intraspecific differences across a latitudinal gradient throughout the Mediterranean Sea.
ACKNOWLEDGEMENTS
The authors are grateful to Melita Peharda Uljević, Vanja Čikeš Keč and Barbara Zorica for their useful advices during sample processing.
FINANCIAL SUPPORT
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
