JOURNAL OF MORPHOLOGY 221:153-160 (1994)

Ultrastructure of the Frontal Sensory Fields in the Lynceidae (Crustacea, Branchiopoda, Laevicaudata)

COBA E. CASH-CLARK AND JOEL W. MARTIN Natural History Museum of Los Angeles County, Los Angeles, California 90007

ABSTRACT The clam shrimp family Lynceidae is unusual in possessing paired fields of short setae on either side of the rostral carina. We describe the position of these fields relative to the direction of water movement in live animals as well as the external and internal structure of these setae. The majority of morphological features support a presumed chemosensory role for these sensilla. These features include the lack of a setal socket and the relatively short length of each seta. The low number of enveloping cells (three or four) is uncharacteristic of chemosensory setae and is more typical of mechanoreceptors, as is the absence of any pores on the setae; these character­ istics indicate that these fields may have both functions, e 1994 Wiley-Liss, Inc.

Branchiopod crustaceans of the family Lyn­ microscopic anatomy by Martin ('92). How­ ceidae have long been recognized as differing ever, ultrastructural details have never been markedly from the four other branchiopod provided. families commonly referred to as "clam Below, we describe the external and inter­ shrimp." Although five families were in the nal ultrastructure of these setae and com­ past united as the branchiopod order Concho- ment briefly on their presumed sensory func­ straca, a grouping that may be revived based tion. on larval development (Clay Sassaman, per­ sonal communication; Martin, '92), morpho­ MATERIALS AND METHODS logical differences between lynceid clam Lynceus gracilicornis were collected from shrimp and clam shrimp of the other four a shallow ephemeral pond in Leon County, families (Cyclestheriidae, Cyzicidae, Leptes- Florida, in April 1984 (see Martin et al., '86 theriidae, and Limnadiidae) are striking for details of the habitat) and again in April (Fryer, '87). These differences have been used 1990, from other ephemeral ponds in north to argue for the separation of lynceids, either Florida. Observations on live specimens were at the level of tribes within the Conchostraca made in 1984 and again in 1990; observa­ (e.g., Linder, '45) or more recently as a sepa­ tions made in 1990 were facilitated by the rate order, the Laevicaudata, with other clam following method. Live animals were re­ shrimp families recognized as the order Spini- moved from water, and the dorsal region of caudata (Fryer, '87; see also Martin and Belk, the carapace valves was quickly dried with '88; Martin, '92). cotton or paper toweling. The dull end of an ()ne of the many unusual morphological insect pin was then glued to the dorsal region features exhibited by lynceids is the posses­ of the valves using a fast-drying waterproof sion of paired, frontal fields of short setae, cement. Pins with clam shrimp attached were located on either side of the midrostral carina mounted in a small lump of modeling clay on just anterior to the location of the eyes. These the edge of a finger bowl containing fresh fields occur in all three genera of the family water, so that the clam shrimp were sus­ {Lynceus, Lynceiopsis, and Paralimnetis; pended in the water directly beneath a stereo- Martin and Belk, '88), and in no other family microscope with drawing tube attached. This of clam shrimp. The function of these fields arrangement allowed positioning the ani­ of setae has never been determined; Martin mals in almost any orientation, so that a et al. ('86), working with the genus Lynceus, clear view of the function of the head and referred to them as sensory fields, and this thoracopods was possible. phraseology has been repeated by Martin and Specimens prepared for scanning electron Belk ('88) and in a review of branchiopod microscopy and transmission electron micros-

e 1994 WILEY4JSS, INC. 154 C.E. CASH-CLARK AND J.W. MARTIN copy were fixed in 3% glutaraldehyde at room and have been commented on by many previ­ temperature in 0.1 M phosphate buffer, post- ous workers (see Martin and Belk, '88). As an fixed in 1% osmium tetroxide an additional 2 example of the size of the fields, each field h, and rinsed in cacodylate buffer before dehy­ measured approximately 260 |xm in width dration in a graded ethanol series. Specimens and 200 \Lxa in length on one animal that had were infiltrated with unaccelerated EM-BED- a head region measuring approximately 2.2 812 embedding medium for 5 days to assure mm in length (measuring the distance from proper infiltration, after which samples were the occipital notch to the tip of the rostrum). infiltrated and embedded with accelerated These fields consist of numerous setae, the EM-BED-812. Gold and silver sections were number of which can vary among individuals cut using a Sorvall MT2-B ultramicrotome, and even between fields on the same animal. stained with uranyl acetate and lead citrate, The number of setae ranges from a low of and examined and photographed using a approximately 50 to perhaps 90 setae per JEOL100CXat80kV. field. Among individuals (n = 8), setal counts were found to differ by as much as from 2 to RESULTS 40 setae. Differences in the number of setae Behavioral observations between the fields of a single individual were less substantial (2 to 15 setae). In contrast to the filter-feeding scenario commonly attributed to all branchiopods and Each seta consists of a long slender shaft, so often depicted in invertebrate texts, lyn- extending from a bulbous base and ending in ceids are active scrapers and scavengers a rather blunt tip. The base of each seta lacks (Fryer and Martin, unpublished observa­ a socket of any type, and is fused to the floor tions; Martin, '92). Although the head is ca­ of the setal field (Figs. 2E, 3B). The length of pable of fitting entirely within the confines of each seta, measured from the base of the seta the closed valves, in life it is more often to the tip, can vary; longer setae may reach a extended forward so that it extends beyond length of approximately 75 ixm while shorter the edges of the carapace valves. This move­ ones may reach a length of only 25 p,m. No ment of the head is accomplished by rotating pore was observed at any point along the the head forward and upward, so that the length of the sensillum. head is bending at approximately the area The cuticular surface underlying these just posterior to the occipital groove (see Fig. fields is thinner than the adjacent cuticle of lA). Whenever swimming forward, the clam the head, and is convexly curved, causing the shrimp's head is therefore positioned such setae to point in various directions (anteri­ that the fields of sensory setae are almost orly, ventrally, and posteriorly) (Fig. 2B-D). perpendicular to the direction of water com­ Internal morphology ing into contact with the head (see arrow, Fig. lA). When food (or food-bearing sub­ Serial cross-sections were cut through the strate such as a small stick or piece of algae) frontal fields to determine if these simple is encountered, the head bends ventrally back setae were innervated. Each seta is inner­ into the region between the valves, so that vated with four dendrites composed of modi­ the labrum and mouthparts are placed in fied cilia (Figs. 3A, 4C), therefore characteriz­ closer proximity to the feeding apparatus, ing the structure as a sensillum. Each which is composed of the maxilla and proxi­ dendrite contains a 9 4- 0 arrangement of mal endites of the thoracopods. microtubules housed within a liquor cavity (=scolopale space) (Fig. 4C). Three (Fig. 3A- D), bly four (Fig. 4B), enveloping cells External morphology surround the ciliary region of the seta. The The head of lynceid clam shrimp is large innermost enveloping cell (el) (=scolopale relative to the body size. In mature individu­ cell) contains the supporting structure, the als it occupies approximately one third of the scolopale, of the ciliary region (Figs. 3A, 4C). total body size and is therefore larger than The scolopale becomes densely packed dis- that possessed by any other clam shrimp tally along the sensillum shaft and produces family. This large head bears two oval fields a thin dendritic sheath, which completely of short, simple setae on either side of the encloses the liquor cavity containing the den­ midrostral carina just anterior (although drites (Fig. 3B). functionally ventral) to the naupliar eye and Within the outer segment of the sensillum rostral pit (Fig. 1B,C). The fields are notice­ (i.e., that portion above the cuticle), the four able even at relatively low magnifications dendrites travel distally for a short distance. SENSORY FIELDS IN LYNCEID CLAM SHRIMP 155

Fig. 1. Lynceus gracilicornis. A: Scanning electron ventral view. Box encloses sensory fields (sf) further micrograph (SEM) lateral view of entire animal, right magnified in C. C: SEM of sensory field, ce, compound valve removed. Arrow indicates range and direction of eye; ne, naupliar eye; p, rostral pit; re, rostral carina. head movement. B: Diagram of entire animal, male, Scale bar in C = 100 |j,m. 156 C.E. CASH-CLARK AND J.W. MARTIN

Fig. 2. Lynceus gracilicomis. SEM micrographs of bar = 100 jim. C: Lateral view of sensory fields. external morphology of sensory fields. A: Frontal view of D: Frontal view of left sensory field. White arrow indi­ female. B: Sensory fields flanking rostral carina and cates rostral pit. E: High magnification of setae within rostral pit. Black arrow indicates rostral pit. Scale sensory fields. Scale bar = 10 jim. Fig. 3. Lynceus gracilicornis. Transmission electron (ds). Scale bar = 2 ixm. D: Cross section at approximately micrographs (TEM) and diagram of single sensory seta mid point of seta. Dendrites (d) becoming disorganized. illustrating internal anatomy. A: Cross section at a level Note three enveloping cells surrounding dendritic sheath below cuticle. Note the four groups of microtubules (mt) (ds). Scale bar = 1 ixm. E: Cross section at level where within the liquor cavity surrounded by scolopale (s). dendrites (d) are highly branched. Note only two envelop­ Scale bar = 1 iJim. B: Cross section at level of bulbous ing cells at this level. Scale bar = 0.05 ixm. F: Cross setal base, where microtubules (mt) begin to adhere to section near tip of seta. Note dendritic sheath and only the ciliary dendritic sheath (ds). Scale bar = 1 iJim. C: one enveloping cell present at this level, c, cuticle; el, Cross section at level where microtubules appear to disap­ enveloping cell 1; e2, enveloping cell 2; e3, enveloping cell pear briefly by closely adhering to the dendritic sheath 3; n, nucleus. Scale bar = 0.05 \im. 158 C.E. CASH-CLARK AND J.W. MARTIN

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Fig. 4. Lynceus gracilicornis. TEM micrographs of B: Cross section of a sensillum illustrating number of sensory setae. A: Cross section of three different sensilla enveloping cells (3). Note (?) represents a possible forth at various levels. Sensillum (a) shows the 9-1-0 arrange­ enveloping cell. Scale bar = 1 fjim. C: Cross section of a ment of microtubules beneath the cuticular surface. Sen­ sensillum at inner segmental level with enlargement of sillum (b) at level where bulbous base fuses to the surface microtubule (m) arrangement surrounded by scolopale of the sensory field. Sensillum (c) sectioned near mid­ (s). Scale bar = 1 fjim. el, enveloping cell 1; e2, envelop­ point. Note position of microtubules. Scale bar = 5 jjim. ing cell 2; e3, enveloping cell 3. SENSORY FIELDS IN LYNCEID CLAM SHRIMP 159 then begin to adhere to the wall of the den­ tors usually terminate at the base of the setal dritic sheath (Fig. 3B) until they appear to shaft and fuse to the inner cuticular wall of disappear briefly (Fig. 3C) and then become the shaft, usually at the level of the movable highly branched or disorganized near the sen- socket; they are thus responsive to move­ sillum midpoint (Fig. 3D-F). This branching ment and vibration. However, such dendrites continues along the length of the setal shaft also can occasionally extend up the canal of within the dendritic sheath and enveloping the shaft of the seta (Altner et al., '83; Derby, cell 1 (el) (Fig. 3P). The ciliary region re­ '89; Felgenhauer, '92). Setae in the sensory mains near the center of the sensillum at all fields of Lynceus do not possess a socket at levels, never coming into contact with the the base, and the dendrites continue distally cuticular wall of the shaft (Fig. 3B-F). Envel­ to the tip of the sensillum and at no point oping cell 2 extends most of the way up the fuse to the wall of the shaft. These character­ shaft but ends just proximal to the apex (Fig. istics would seem to argue for a chemorecep- 3E), while enveloping cell 3 (e3) appears to tive function. Additionally, the scolopale of end near the midlength point of each seta mechanoreceptors is thought to produce a (Fig. 3D,E). The cuticle covering the sensil­ very thick dendritic sheath, whereas in che­ lum appears rather thin at the distal region moreceptors they produce a thin dendritic of the sensillum, as opposed to the thicker sheath (Felgenhauer, '92); the relatively thin proximal region. dendritic sheath in Lynceus again would agree Figure 4A illustrates a section cut through with the ultrastructure most typical of chemo­ the convexly curved sensory field, which al­ receptors. lows a detailed look at three different sensilla However, some features are not in agree­ at various levels. Sensillum (a) was cut at a ment with the usual depiction of the typical more proximal level beneath the cuticle (in­ crustacean chemoreceptive sensillum. The sa­ ner segment), as is recognizable by the four lient exceptions are the number of envelop­ dendrites organized in the 9 -f- 0 arrange­ ing cells that surrounds the dendrites and ment surrounded by the scolopale (see also the absence of a pore. We observed three or Fig. 4C). Sensillum (b) was cut at the level in four enveloping cells, as did Crouau ('89) which the cuticular floor of the setal field when describing chemosensitive dendrites in fuses to the cuticle of the setal shaft and mysidaceans, which is a number more typical clearly demonstrates the absence of any setal of mechanoreceptors, although the number socket. Sensillum (c) was cut along the mid of enveloping cells is known to vary (Guse, region as the microtubules appear to disap­ '78, '80). The absence of a pore is usually a pear while closely adhering to the dendritic feature of mechanoreceptors, although, as sheath. Laverack and Barrientos ('85) note, the mode DISCUSSION of entry of chemical stimulants may be by direct diffusion across the thin cuticle of the The setal fields of Lynceus have been de­ sensillum; absence of any obvious pore does scribed in previous papers as probably having not preclude the possibility of chemosensory a sensory function (Martin et al, '86; Martin function. Ache ('82) also found that chemo­ and Belk, '88; Martin, '92). The present ultra- sensory setae of decapod aesthetascs possess structural study supports the idea that these a permeable tip rather than a pore. Both fields are sensory in nature and at least dem­ studies (Ache, '82; Laverack and Barrientos, onstrates the fact that they are innervated. *85) found that stimulants reached the inter­ Further classification of the setae as being nal dendrites, despite the absence of any obvi­ either chemo- or mechano-receptors is more ous pore in the integument of the setal shaft, difficult. and this may be the mode of chemical entry According to Felgenhauer ('92), both into the sensory setae of Lynceus as well. mechanoreceptors and chemoreceptors have similar basic microanatomy, although mor­ External morphological characteristics of phological differences between the two may crustacean sensilla are also in support of a sometimes allow workers to elucidate their predominantly chemosensory role of these function. Mechanoreceptors usually feature setae in Lynceus gracilicornis. Typical crusta­ a movable socket at the base of the sensillum cean chemoreceptive sensilla are short, not (e.g., Altner et al., '83; Hamilton et al., '85; robust or thick, often occur in numerous Felgenhauer and Abele, '83; Felgenhauer, quantities, and are unlikely to be sensitive to '92; Laverack and Barrientos, '85). The den­ mechanical disturbance (Laverack and Barri­ drites of the outer segment of mechanorecep­ entos, '85). Scanning electron microscopy il- 160 C.E. CASH-CLAHK AND J.W. MAHTIN lustrates these features for the frontal sen­ ing in Crustacea. Crustacean Issues, Vol. 6. Rotterdam, sory fields ofLynceus and further argues for Netherlands; A.A. Balkema Press, pp. 27-47. Felgenhauer, E. (1992) Chapter 2: External Anatomy their primary function being one of chemore- and Integumentary Structures. In F. Harrison and A. ception, although duality of function (chemo- Humes (eds): Microscopic Anatomy of Invertebrates, and mechano-reception) is not unprecedented Vol. 10: Decapod Crustacea. New York: Wiley-Liss, in crustaceans and may be occurring here as Inc., pp. 7-^13. Felgenhauer, B.E., and L.G. Abele (1983) Ultrastructure well. and functional morphology of feeding and associated appendages in the tropical fresh-water shrimp Atya ACKNOWLEDGMENTS innocous (Herbst) with notes on its Ecology. J. Crusta­ The authors thank Dr. Bruce Felgenhauer cean Biol. 3:336-363. for providing information and comments on Fryer, G. (1987) A new classification of the branchiopod Crustacea. Zool. J. Linn. Soc. 9I.-357^383. an earlier draft. We also thank Alicia Thomp­ Guse, G.W. (1978) Antennal senilla of Neomysis integer son for her assistance with transmission elec­ (Leach). Protoplasma 95:45-161. tron microscopy at the University of South­ Guse, G.W. (1980) Development of antennal sensilla dur­ ern California Electron Microscopy Center. ing moulting in Neomysis integer (Leach) (Crustacea, Funds were provided by NSF grant number Mysidacea). Protoplasma 105:53-67, Hamilton, K.A., K.A. Linberg, and J,F. Case (1985) Struc­ BSR 9020088 to J.W. Martin. ture of dactyl sensilla in the kelp crab, Pugettia pro- diicta. J. Morphol. iS5;349-366. LITERATURE CITED Laverack, M.S., and Y. Barrientos (1985) Sensory and Ache, B.W. (1982) Chapter 8. Chemoreception and ther- other superficial structures in living marine Crustacea. moreception. In H.L. Atwood and D.C. Sandeman (eds): Trans. R, Soc. Edinb. 76:123-136. The Biology of Crustacea, Vol. 3, Neurobiology: Struc- Linder, F. (1945) Affinities within the Branchiopoda, ture and Function. New York: Academic Press, pp. with notes on some dubious fossils. Arkiv Zool. 37:1- 369^398. 28. Altner, I., H. Hatt, and H. Altner (1983) Structural Martin, J.W. (1992) Chapter 3: Branchiopoda. In F. Har­ properties of bimodal chemo- and mechanosensitive rison and A. Humes (eds): Microscopic Anatomy of setae on the pereiopod chelae of the crayfish, Austropot- Invertebrates. Volume 9: Crustacea. New York: Wiley- amobius torrentium. Cell Tissue Res. 223.-357~374. Liss, Inc., pp. 25-224. Crouau, Y. (1989) Feeding mechanisms of the Mysidacea. Martin, J.W., and D. Belk (1988) Review of the clam In B.E. Felgenhauer, L. Wathng, and A.B. Thistle shrimp family Lynceidae Stebbing, 1902 (Branchiopoda: (eds): Functional Morphology of Feeding and Groom­ Conchostraca), in the Americas. J. Crustacean Biol. ing in Crustacea. Crustacean Issues, Vol, 6. Rotterdam, 8:451-482. Netherlands: A.A. Balkema Press, pp. 27^7. Martin, J.W., B.E, Felgenhauer, and L.G. Abele (1986) Derby, CD. (1989) Physiology of sensory neurons in Redescription of the clam shrimp Lynceus gracilicarnis morphologically identified cuticular sensilla of crusta­ (Packard) (Branchiopoda, Conchostraca, Lynceidae) ceans. In B.E. Felgenhauer, L. Watling, and A.B. Thistle from Florida, with notes on its biology, Zool. Scr. (eds): Functional Morphology of Feeding and Groom­ 15:221-232,