Diversity and arrangement of the cuticular structures of Hyalella ( Crustacea : Amphipoda : Dogielinotidae ) and their use in taxonomy

This study describes the morphology and arrangement of the cuticular structures of Hyalella castroi Gonzalez, Bond-Buckup & Araujo, 2006 and Hyalella pleoacuta Gonzalez, Bond-Buckup & Araujo, 2006, to identify specific characters that can be used in taxonomstudies of this genus. The entire cuticular surface of both species was examined by optical and scanning electron microscopy. The data obtained were compared with available information for other members of Peracarida, mainly Amphipoda and Isopoda. Five different types of cuticular structures, including 30 types of setae, four types of microtrichs, three types of pores, and some structures formed by setules and denticles were identified. The results were compared with other groups of gammarids, and peracarideans, such as Thermosbaenacea and Isopoda. The use of cuticular structures as a tool for taxonomic studies showed important results, not only at species level, but also at genus, and family levels.

continental waters of the Americas, where they constitute important links in the food chains, serving as a food resource for aquatic birds, fish, and other crustaceans (GROSSO & PERALTA 1999).This genus is morphologically quite diverse, mainly in South America (GONZÁLEZ et al. 2006), although some species have a very similar morphology that makes their differentiation and identification difficult.
In view of the scarcity of information of cuticular structures of Amphipoda and more precisely within species of the genus Hyalella, we analyzed the morphology and arrangement of the cuticular structures, of Hyalella castroi González, Bond-Buckup & Araujo, 2006 and Hyalella pleoacuta González, Bond-Buckup & Araujo, 2006.

MATERIAL AND METHODS
Specimens of H. castroi and H. pleoacuta were collected in fishponds near the source of the Rio das Antas, at the Vale das Trutas, Municipality of São José dos Ausentes, state of Rio Grande do Sul, Brazil (28º47'00"S, 49º50'53"W).Thirty adult specimens of both sexes of each species were kept in 500 ml beakers filled with distilled water, without food, for three days, in order to improve the cleanliness of the appendices.They were then fixed in 70% ethanol and dissected under a stereomicroscope.For the SEM analyses, the dissected appendages, together with four whole females and four whole males of each species were prepared according to the technique of LEISTIKOW & ARAUJO (2001).The material was examined in a Jeol JSM 6060 scanning electron microscope (SEM) of the Microscopy Center of the Universidade Federal do Rio Grande do Sul, operated at 10 Kv.Part of the dissected appendages were mounted on slides ZOOLOGIA 26 (1): 127-142, March, 2009 in liquid glycerin under coverslips, and observed in an Olympus CX 31 microscope fitted with a drawing tube for observation of the internal morphology of the setae.The general description of each appendage follows GONZÁLEZ et al. (2006).
Up to the present, none of the classification schemes proposed for the cuticular structures of crustaceans has been able of embracing the full diversity of these structures among the group.The majority of these studies were conceived with an emphasis on only one type of structure, such as setae (THOMAS 1970, FISH 1972, FARMER 1974, OSHELL & STEELE 1988) or microtrichs (OSHEL et al. 1988).For this reason, in the present study a more inclusive classification was developed, including all of the diversity found in the two species of Hyalella worked here unified to the nomenclature of these structures found in the literature, facilitating their use in future comparative studies.
To this end we opted to combine preexisting schemes, preferentially those that were developed based on data from electron microscopy.For the definitions of seta, setule, and denticle we employed the proposal of GARM (2004a).However, for didactic reasons, only the setules and denticles that issue directly from the surface of the cuticle were considered as cuticular structures.These same structures, when present on the setal shaft were considered as a character of the setae and were described as such.
The terminology used to describe the setae followed WATLING (1989).To this terminology we added the term lamella, sensu CALAZANS & INGLE (1998), to describe structures of the setal shaft.The term "sensory spine" sensu BRANDT (1988) was re-placed by the term "cuspidate seta with accessory seta" as advocated in the definition of a seta by GARM (2004a).Microtrich was identified according to the proposal of OSHEL et al. (1988).
All the setae were identified with a letter that indicates their category (A-G), and a number that indicates the number of variations found (FACTOR 1978, COELHO & RODRIGUES 2001a, b).The pores were named according to their specific morphology.Setules (S) and the polygonal patterns described for pores (P) and denticles (T) were identified by a letter that indicates the nature of the structure that composes it, and a number corresponding to the number of variations found for these structures.Each description was illustrated with a SEM micrography.
As the terms microtrichs, setules, and denticles are not used in a uniform way in the crustacean literature, table I presents the comparisons between the terms used here and those used in other crustacean studies.
The setae were the most abundant and diverse structures found on the cuticular surface.Altogether, 30 variations of setae were observed , that were allocated to seven groups: simple, cuspidate, plumose, pappose, serrulate, serrate, and pappo-serrate (Tab.II).The table III shows the comparisons between the setae described in this study and those of other members of Peracarida.Lamellate seta: shaft smooth, of varying length, lamellate tip varying in length, occupying between 1/2 and 1/5 of the terminal portion of the shaft, with a terminal pore (Fig. 5).
Antennae, maxillipeds, gnathopods and pereopods A2 Shaft long and smooth, of similar diameter along its entire length, with annulation and terminal pore (Fig. 6).

A6
Curved seta: shaft long and smooth, annulation marked and subterminal pore with lamellate tip.Distal end of shaft curved and decorated with little.

B4
Shaft variable in size, with wide base gradually tapering to apex, terminal pore on accessory seta inserted on final third of shaft.Articulation commashaped (Fig. 18).

Palm of gnathopod 2 of males B5
Shaft short with wide base, slightly concave, tapering gradually to end, terminal pore on accessory seta inserted in final third of shaft on opposite side to concavity.Articulation wide (Fig. 19).
Anterior lobe of gnathopods B6 Very similar to type B4, but with variable length, and simple, round articulation (Fig. 20).

D2
Shaft long and robust with proximal half smooth; distal half of shaft with three rows of long setae (Fig. 24).
Inner plate of maxilla 2

D3
Shaft short, with setules on distal half of shaft, arranged in several rows grouped on one side of the shaft.On distal third, the setules are arranged around shaft (Fig. 25).
Ventral inner border of inner plate of maxilliped D4 Shaft long, with setules arranged randomly from basal.Annulation weak (Fig. 26).

E1
Shaft long, with wide setules arranged in two opposite rows on distal two thirds.Annulation present (Fig. 27).
Inner plate of maxilla 2 E2 Shaft long and slender, with short slender setules arranged in two opposite rows from distal half of shaft.Annulation weak (Fig. 28).
Inner plate of maxilla 2

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Only type I microtrichs (sensu OSHEL et al. 1988) were found in the two species of Hyalella, with four subtypes identified (Tab.IV, Figs 35-39).Subtype Id is described here for the first time.Type II microtrichs are absent in both species.The microtrichs are observed on antennae, mouthparts, gnathopods, pereopods and on dorsal surface.
The setules showed two variations (Tab.IV), one that occurs on the inner surface of the oostegites (Fig. 40) and another on the mouthparts (Fig. 41).
We also observed three types of pores: simple, knobbed and projected (Tab.IV, Figs 42 and 43).The first two occur on the entire cuticular surface, whereas the last occurs only on the surface of the mouthparts.In some areas of the cuticle, the simple and knobbed pores were arranged in small polygons bounded by a narrow bar of non-porous cuticle.In general, several of these polygons were grouped together, forming what was termed by BRADBURY et al. (1998) as "polygonal patterns" Two distinct patterns of distribution of the pores were observed within these polygons (Tab.IV, Figs 43 and 44).
Denticles were found mainly on the gnathopods and pereopods.These structures did not show significant variations in their morphology and generaly were grouped in two ways, forming either polygonal patterns (Fig. 45) or a comb scale (Fig.

46, Tab. IV).
Hyalella castroi and H. pleoacuta are very similar in respect to type, morphology, and arrangement of the cuticular structures (Figs 47-75).However, differences were observed in the number of setae on the appendages and between the structures of the gnathopods of males and females in both species.Table V shows the arrangement of the structures on the appendages of the two species.

Diversity of cuticular structures Setae
In both H. castroi and H. pleoacuta, three distinct types of cuspidate setae with an accessory seta were observed: B4, B5,   29).
Outer plate of maxilla 2 F2 Similar to type F1, but on distal one third, of the side opposite to the denticles, there is a row of short setules; subterminal pore (Fig. 30).
Outer plate of maxilla 2 F3 Shaft variable in length, robust, with terminal pore on lamellate and curved tip.Denticles in two nearly opposite rows on distal half of shaft.Annulation present (Fig. 31).
Peduncle of antennae, palp of maxilliped and gnathopods   and B6 .In other peracaridans such as the amphipod G. pseudolimnaeus and the isopod E. pulchra, only two types were recorded (BRANDT 1988, READ & WILLIAMS 1991).The seta B4 (Fig. 18), which has a movable socket, until the present was observed only in Hyalella males.The other two setae, B5 and B6, have a similar morphology to those observed in G. pseudolimnaeus and E. pulchra, but differ from these mainly in their arrangement on the appendages and the ornamentation of the accessory seta (Tab.VI).The cuspidate seta with accessory seta is, up to the present, exclusive to the Peracarida, and its morphology, ornamentation, and arrangement can characterize families, genera, or even species (BRANDT 1988).Our data, together with the above information, show that the arrangement of the cuspidate setae with accessory seta and the ornamentation of the accessory seta constitute a genus character for Hyalella.It is also worthy of mention that the analysis of these structures must take into account both aspects, morphology and arrangement, as the combination of them constitutes a genus characteristic.
In Decapoda, the articulation of plumose setae is always supracuticular (GARM 2004a, b).However, in Peracarida these setae can show two types of articulation, infra or supracuticular, as observed in Thermosbaenacea by WAGNER (1994).In Hyalella, the plumose setae always have an infracuticular articulation (Fig. 21), a characteristic also observed in the lotic amphipod G. pseudolimnaeus (READ & WILLIAMS 1991), in the marine amphipod Gammaropsis inaequistylis (Shoemaker, 1930) and Hyale nilsoni Hatke, 1843 (OSHEL & STEELE 1988), and also in the intertidal isopod E. pulchra (FISH 1972).Comparison of our data with available information in the literature indicates that the type of infracuticular articulation of the plumose setae is a character shared between amphipods and isopods.A close relationship between these two taxa was proposed by POORE (2005), and is now corroborated by this character.

Microtrichs
Differing from what were observed for the majority of amphipods by LAVERACK & BARRIENTOS (1985), OSHEL et al. (1988), andOLYSLAGER &WILLIAMS (1993), and also for some isopods Simple Simple and rouded pores on three sizes: small, medium, and large.
Covered the surface of body Knobbed Medium-sized pores with a knob on one side.

Covered the surface of body
Projected They have a tube-shaped prolongation so that the pore opening is above the cuticular surface (Fig. 42).Mouthparts Pores "polygonal patterns" (P) (Figs 43-44)

P1
Each polygon has several "knobbed" pores randomly arranged, with small simple pores between them.One large simple pore is present at some points where the polygons converge (Fig. 43).

P2
Similar to P1, but only the simple middle pores are present (Fig. 44).

Denticles (T) (Figs 45-46)
T1 Polygonal pattern: the denticles are arranged in increasing, crescentic rows, and the inner part is filled by smaller denticles, forming a geometric pattern similar to a polygon.Several of these fit side by side and cover large areas on some appendages (Fig. 45).
Uper lip and gnathopods T2 Comb scale: the denticles are arranged in increasing straight rows or in crescents with no inner filling.Each row of these denticles has a united base that may be raised above the cuticle, forming a scale-like structure (Fig. 46).

Setules
The record of setules S1 occurring on the inner surface of the oostegites of both species of Hyalella is new for Peracarida.However, their morphology corresponds to that of the "barbed seta" found by WAGNER & BLINN (1987) on the maxilla of H. azteca and H. montezuma.

Pores
Little is known about the arrangement of pores on the cuticular surface of Amphipoda, but it's known that these are abundant and show quite varied arrangements (HALCROW & BOUSFIELD 1987).In H. castroi and H. pleoacuta we observed two different patterns of pore arrangement, always within polygons.Each pattern is found in a specific area of the cuticle, with an identical arrangement in both species.Comparing these patterns with those described for other gammarideans by HALCROW & BOUSFIELD (1987), we percive that pattern P1 (Fig. 42) is identical in form as well as arrangement on the cuticle, to that observed for another dogielinotid, Proboscinotus loquax (Barnard, 1967).Pattern P2 (Fig. 43), although identical in form to that observed for Eohaustorius washingtonianus (Thorsteinson, 1941), differs from this in its arrangement on the cuticle.None of the patterns found here is comparable to that observed by READ & WILLIAMS (1991) for the freshwater gammarid G. pseudo-  23) pappose seta D1 from antenna 2; (24) pappose seta D2 from inner plate of maxilla 2; (25) pappose seta D3 from inner plate of maxillipeds; (26) pappose seta D4 from inner plate of maxillipeds; (27) serrulate seta E1 from inner plate of maxilla 2; (28) serrulate seta E2 from inner plate of maxilla 2; 29) serrate seta F1 from outer plate of maxilla 2; (30) serrate seta F2 from inner plate of maxilla 2; (31) serrate seta F3 from gnathopod 2; (32) serrated seta F4 from maxilla 1; (33) papposerrate setae G1 from inner plate of maxilla 2; (34) papposerrate setae G2 from inner plate of maxilla 1. limnaeus, which suggests that this characteristic is not associated with the environment.According to HALCROW & BOUSFIELD (1987) and HALCROW & POWELL (1992), the arrangement of pores on the cuticular surface of Amphipoda is a family-level character.This hypothesis is corroborated by our data, and at the same time reinforces the proposal of SEREJO ( 2004), who recently transferred the genus Hyalella to the family Dogielinotidae.Further, with respect to the pores, we observed that some material is expelled through the larger pores (Fig. 43), as previously suggested by HALCROW (1978HALCROW ( , 1985)), BOROWSKI (1985), MOORE &FRANCIS (1985), andHALCROW &BOUSFIELD (1987).

Denticles
The polygonal patterns (Fig. 45) and the comb scales (Fig. 46) formed by the denticles have been reported for gammaridean amphipods by many workers as "polygonal pattern" WILLIAMS & BARNARD (1988); "echinate fields" sensu HOLMQUIST (1989); "rugosities" sensu BRADBURY et al. (1998); "scutellated scales" and "cae-spitose patch" sensu JAUME & CHRISTENSON (2001).In adition, comb scale-like structures were also reported for some isopods "microtrich crescentic" sensu NEEDHAM (1942) andFISH (1972).Both types of comb scale-like structures were used by WILLIAMS & BARNARD (1988) in characterizing the freshwater families Neoniphargidae and Crangonyctidae.However, comparisons between literature data and our results are difficult because the avaiable descriptions and photographs are not detailed and there is no consensus in the use of terminology.BRADBURY et al. (1998), working with different families of marine gammarideans, stated that their "rugosities", located basically on both gnathopods, are produced by microsetae, that is, structures that show a point of articulation with the cuticle; however, in some of his figures, it is clear that these rugosities are denticles.The "echinate fields" of Talitroides alluaudi (Chevreux, 1896) and Talitroides topitotum (Burt, 1934) (HOLMQUIST 1989) are actually produced by small setae, and because of this cannot be compared to the formations observed here.Similarly, the cuticular polygons cited by WILLIAMS & BARNARD (1988) were observed only with a light microscope, which makes it difficult to define the kind of cuticule structure that compose them.For the genus Hyalella, this cuticular structures and its arrangement on the appendages were shown to be important characters (see discussion below) for separation of the species.

Location of cuticular structures Mouthparts
Examination of the mouthparts of Hyalella species (Figs 49-62) revealed great similarity in form as well as in structure.The setae found on maxillas 1 and 2 of the two species did not differ, at least in general morphology and diversity, from those observed by WAGNER & BLINN (1987) for H. azteca and H. montezuma.This was expected, because the morphology of the mouthparts differs little among related species of Amphipoda (ARNDT et al. 2005).However, some intra-and interspecific variability in the type of structure (setules and/or denticles) as well as in their distributional pattern  was observed in the ornamentation of the ventro-proximal surface of the lower lip.
Similar to observed in Thalassinidea by COELHO & RODRIGUES (2001a, b) and PINN et al. (1999) a wide diversity of setal types was found in the mouthparts of the two species, indicating that they are capable of manipulating a wide variety of food items and can use more than one feeding mode (MACNEIL et al. 1997, ARNDT et al. 2005).When more than one feeding modes are possible, the principal mode that the animal uses to obtain food can vary with the microhabitat and its available resources.From the ecological perspective, this character constitutes a great adaptive advantage for this genus.
A peculiar character of the mouthparts of both species is the presence of elongated pores.Similar pores, called excretory pores, were also found in Lophogaster typicus M. Sars, 1857 by DE JONG et al. (2002), andin Isopoda (GORVETT 1946).These pores probably function to lubricate food particles, as suggested for the shrimp Penaeus merguiensis De Man, 1888(MCKENZIE & ALEXANDER 1989).The arrangement of pores on the mouthparts is different in all the species and groups previously mentioned; however, the sparse information on the subject does not allow us to evaluate its taxonomic value.

Other appendages
On the other appendages, the most significant differences between the two species were observed in the ornamentation of gnathopods 1 and 2. The distal end of the both gnathopods The distribution of B6 on uropod 1 and the number of cuspidate setae on the telson were also considered a species character for Hyalella.
The morphology, arrangement, and diversity of cuticular structures of Hyalella constitute important tools for taxonomic analyses, principally at the genus and species levels.Moreover, the comparisons made here provide strong indica-tions that cuticular structures can contribute very significantly to elucidate the systematics of Peracarida.

Plumose
, with setules densely arranged in two rows along entire lenght.(Fig.21).PleopodsC2Shaft long, with setules beginning after the annulation.These setules usually roll around their own axis, forming loops on the sides of the shaft (annulation marked, distal half of shaft branched in long setules with smooth edges, forming a tuft.(Fig.23).

Table I .
Cuticular structures found in the present study for H. castroi and H. pleoacuta compared with other crustaceans with data from

Table II .
Description and distribution of setal types on the cuticular surface of H. castroi and H. pleoacuta.

Table II .
Continued.

Table IV .
Description and distribution of microstructures on cuticular surface of H. castroi and H. pleoacuta.IaShaft short, with terminal pore directed to one side, and lamellas decorating the shaft on the side opposite the opening of the pore.On this same side, a hood projects apically, covering the pore (variation of type Ia ofOSHEL et al. 1988) (Fig.35).