Gill-Cleaning Mechanisms of a Dendrobranchiate Shrimp, Rimapenaeus Similis (Decapoda, Penaeidae): Description and Experimental Testing of Function

JOURNAL Op MORPHOLOGY 2-12:125-139 (19991

Gill-Cleaning Mechanisms of a Dendrobranchiate Shrimp, Rimapenaeus similis (Decapoda, Penaeidae): Description and Experimental Testing of Function

RAYMOND T. BAUER* Department of Biology, University of Louisiana at Lafayette, Lafayette. Louisiana

ABSTRACT Observations on functional morphology and results from experi­ ments demonstrate that setiferous epipods compose the major gill-cleaning mechanism in a penaeoid shrimp, Rimapenaeus similis. Epipods on the second maxillipeds and on pereopods 1—3 are equipped with long setae bearing an array of digitate scale setules. These multidenticulate setae reach to most gills and are jostled among them during limb movements. Experi­ ments were performed in which epipods were removed from the gill chamber on one side (experimental) but not the other (control); treated animals were exposed to fouling in a recirculating water system for 2 weeks. Particulate fouling, measured by reduction in relative gill transparency, was significantly greater on experimental than control gills. The pereopodal exopods, not previously implicated in gill cleaning in any decapod, were similarly identified as important gill-cleaning structures. Equipped with long multidenticulate setae like those on the epipods, exopods sweep back and forth over the gill filaments just under the gill cover, areas not reached by the epipods. Exopod- ablation experiments were conducted that showed that exopods prevent particulate fouling on gill surfaces over which they sweep. The similarity in action of the passive gill-cleaning system of R. similis to that of crayfish (Bauer [1998] Invert Biol 117:29-143) suggests the hypothesis that the epipodal and exopodal cleaning setae of R. similis are ineffective against epibionts. The reduction in epipodal and exopodal cleaning systems that occurs in the Penaeoidea is hypothesized to be compensated for by increased development of gill-cleaning setae on the branchiostegite, scaphognathite, or other structures. J. Morphol. 242:125-139,1999. e< 1999 Wiley-Liss, Inc. KEY WORDS: cleaning; epipods; exopods; fouling; gills: shrimp

One of the unique features of decapod conditions for the settlement and growth of crustaceans (e.g., shrimps, lobsters, crabs) is microfouling organisms such as epibiotic bac­ that the gills are enclosed in a branchial teria and protistans. As a result, most deca­ chamber. The advantages of gill enclosure pods show adaptations for keeping gills are that the gills are protected from mechani­ free of fouling that accumulates between cal injury and, in the narrow confines of a molts. In many caridean and stenopodidean branchial chamber, water can be pumped shrimps, as well as anomuran crabs and rapidly over gill filaments. However, there is callianassid "ghost shrimps," gills are cleaned a disadvantage to gill enclosure: the mass of by cheliped brushing. Specialized chelipeds gill filaments in a restricted space serves as with brushes of complex setae are inserted a sediment trap (Bauer, 79, '89, '98). Particu­ late matter earned in by the inhalant water flow may be caught by or settle on gill fila­ Contract grant sponsor: NOAA Louisiana; Contract grant num­ ments, covering their surfaces and prevent­ ber: Sea Grant NA89AA-D-S266: Contract grant sponsor: the ing the gill functions of gas exchange, excre­ Noptune Trust. ^Correspondence to: Raymond T. Bauer, Department of Biol­ tion, and ion regulation. The rapid flow of ogy, University of Louisiana at Lafayette, Lafayette, LA 70504 - water past the gills also creates favorable 2451. L'-nmil: [email protected]

0 1999 WII.BY-L1SS. INC. 126 R.T. BAUER into the gill chamber and the decapod ac­ MATERIALS AND METHODS tively brushes and picks fouling material Rimapenaeus similis (Smith, 1885) is a from the gills (Bauer, '79, '81, '89). The effi­ penaeid species formerly included in the ge­ ciency of cheliped brushing in keeping gills nus Trachypenaeus s.L, revised by Perez free of both epibiotic and particulate fouling Farfante and Kensley C97). Specimens were has been demonstrated experimentally by collected by otter trawl in Mississippi Sound Bauer ('79) in caridean shrimp and Pohle near Horn Island (30° 15'N, 88° 45'W) and ('89) in an anoinuran crab. the Pascagoula Ship Channel (30° 20'N, 88° In other decapods, no such active gill- 32'W), Mississippi, on several trips during cleaning mechanism has been observed. 1991-1993. Live specimens used in observa­ However, there, appear to be indirect or pas­ tions and experiments were maintained indi­ sive methods for cleaning gills in these vidually on recirculating water tables with groups. Setobranch setae arise from the coxal seawater at 25-30 ppt, water temperatures (most proximal) segment of the thoracic legs of 20-25°C, a 14/10hr day:night schedule, and and are directed on and among the gill fila­ daily feeding with commercial shrimp food ments. Upon movements of the thoracic in pellet form. Specimens used in morpho­ limbs, these setae are jostled and thrust logical work were initially fixed in 10-15% among the filaments, presumably cleaning seawater formalin, later washed with water, them. Setobranch setae are found in many taken through a series of washes of 25%, caridean shrimps, thalassinid and axiid 35%, and 50% ethanol up to final storage in thalassinideans, and in parastacoidean and 70% ethanol. Material used for scanning elec­ astacoidean crayfishes. In other decapods, tron microscopy was taken through a gradu­ coxal outgrowths (epipods) positioned be­ ated series from 70-100% ethanol, dried in tween gills bear' setae similar in microstruc- C02 with an EMS 850 critical-point dryer, ture to setobranch setae and to those in and sputter-coated with gold-palladium for cheliped gill brushes. These setiferous epi­ 1-8 min at 10—20 nm/min, with longer coat­ pods, found in penaeoidean shrimps, palinu- ing times used for larger, topographically rid and nephropid lobsters, and brachyuran complex samples. Specimens were viewed crabs have been presumed to be passive with a JEOL 7000 FV scanning electron gill-cleaning structures (Bauer, '89). Batang microscope at an accelerating voltage of 15 and Suzuki C99) have recently described both kv. Perez Farfante and Kensley ('97), Perez setobranchs and setiferous epipods in the Farfante (71), aud Dall ('57) were followed "mud lobster" Thalassina anomala. Other in designation of gill type in R. similis. indirect gill-cleaning methods include multi- Observations were made on living Rimap­ denticulate setae fringing the scaphogna- enaeus similis in order to observe grooming thite (gill bailer) in some decapods (Bauer, behaviors, especially possible brushing of '79; Suzuki and McLay, '98), as well as simi­ gills by chelipeds or other appendages. Speci­ lar setae on the inner side of the branchio- mens were set up in recirculating aquaria stegite (gill cover) in cambarid crayfishes with sand substrates. A total of 23 individu­ (Bauer, '98). The only experimental study of als were observed during the day for dura­ the actual function of putative indirect gill- tions of 0.5-1.0 hr, for a total of 19 hr. Addi­ cleaning mechanisms was performed on the tional observations were taken from crayfish Procambarus clarkii (Bauer, '98). recordings made at night, when R. similis is Setobranch and branchiostegal setae were most active, with a low-light, infrared-sensi­ shown to be very efficient at preventing sedi­ tive surveillance video camera, both at real ment fouling, but they were not effective time speed and a time-lapse speed of 10 against microbial fouling. pictures/sec (12-hr period on a single VHS videocassette), for a total of 90 hr on 17 individuals. Light for night recordings was The gill-cleaning mechanisms of a major supplied by infrared lamps (880 nm). decapod crustacean group, the Penaeoidea, The possible gill-cleaning function of epi­ have only been suggested (Young, '59; Bauer, pods and exopods was tested by removing '81, '89; Dall et al., '90) and have never been them, in separate experiments, from the demonstrated experimentally. The objective branchial chamber of one side of the shrimp of this study was to identify, to describe, and but not the other. First attempts at ablation to test experimentally possible gill-cleaning experiments were interrupted by molting of mechanisms in a member of this group, the treated animals within a few days to a week penaeid shrimp Rimapenaeus similis. of ablation. This problem was reduced by GILL-CLEANING MECHANISMS OF R. SIMIL1S 127 performing the ablations 1-4 days after the individuals could be ablated on a single day. molt of individually maintained shrimps, i.e., Shrimps were anesthetized by temperature sufficient time for a shrimp to recover from a shock before ablations by changing them molt, but long enough so that several molted from water of ambient temperatures of 21-

Fig. 1. Rimapenaeus similis A: Anterior end of of pereopod 4; e, gill-cleaningepipod; mle, epipod of lirst. shrimp, with branchiostegite or gill cover (dotted line) maxil.iped; m2, maxilliped 2; m3, maxillipcd 3; pa, pos- cut away to reveal gills. Area outlined by box is shown at terior arthrobranch; pd, podobranch; pl-p5, pereopods higher magnification in B and Figure 2A,B. B: Area of 1-5 (arrows point to basi-ischial breakage plane, with gill chamber outlined by box in A, showing exterior view distal portion of limb removed); s. scaphognathite (gill of undisturbed, complete set of gills, exopods, and cpi- bailer); x, pcreopodol exopod. pods. aa. anterior arthrobranch; ar, single arthrobranch 128 R.T. BAUER 23°C to 12-15°C. Epipods or exopods were TABLE 1. Distribution of gills, epipods. and exopods in removed from the right side of anesthetized Hi mapenaeus similis shrimps using fine forceps. Treated shrimps Bodv Somite 7 8 9 10 11 12 13 14 were then maintained individually on water Thoracic Somite 1 2 3 4 5 6 7 8 tables and exposed to fouling for 14 days, Limb Ml M2 M3 n P2 P3 P4 P5 after which they were anesthetized by chill­ Pleurebranchs 0 0 1 I 1 I 0 0 ing and then preserved and stored in 10- Arthrobranehs r 2 2 2 2 1 0 15% buffered seawater formalin. Podobranchs 0 1 0 0 0 0 0 0 Epipods lb lc 0 lc lc lc 0 0 Gill fouling was measured in 25 individu­ Exopods H It' If lc lc lc lc V als randomly selected from those that had b, broad blade, fringed with short, plumose setae; c, equipped survived the 14-day duration of the experi­ with lorig setae studded with digitate scale setules. cleaning hypoth­ ment without molting (all 35 after epipod esized as principal function; f, flagelliform. equipped with plu­ ablation; in the exopod ablation, 9 of 42 mose setae; M. maxilliped: P, pereopod; r, rudimentary gill com­ molted 1-2 days before the end of the experi­ posed ol'small number of filaments; v = reduced in size, vestigial ment). Particulate fouling of a gill was quan­ tified by measuring its relative transpar­ maxilliped 2, the posterior arthrobranehs of ency when mounted on a slide in a compound maxilliped 3 and pereopods 1-2, and the light microscope, as in Bauer ('98). A gill was pleurobranchs of maxilliped 3 and pereo­ mounted in water on a depression slide and pods 1-3 (Figs. 2A, 3B). covered with a cover slip. Standardized loca­ tions on a gill were viewed at. 250x so that, gill Setiferous epipods have been hypothesized tissue completely filled the field of view. For as gilldenning structures in penacid shrimps the epipod-ablation experiment, all gills were (Young, '59 ; Bauer, '81, '89; Dall et al., '90). examined from the third maxillipeds and third In Rimapenaeus similis, such epipods are pereopods, located anteriorly and posteri­ found on maxilliped 2 and pereopods 1-3 orly, respectively, in the gill chamber. Mea­ (Figs. IB, 2A,B, 3A-E, 4). Each arises from the sures of transparency were taken midway coxal segment as a narrow horizontal strap, along the length of the gill. In the exopod-abla- expanding into a large, thin blade that ex­ tion experiments, readings were taken on tends up among the gills (Figs. 3B-D, 4). the anterior arthrobranehs of the third max­ The epipod blades are wide proximally but illipeds and pereopods 1-3, halfway between are slightly narrower distally, and bifurcate the midpoint and proximal end of each gill. into narrow branches in pereopods 1 and 2 but continue without interruption in pere­ Intensity of light passing through the gills opod 3 and maxilliped 2 (Fig. 4). Epipods of was measured with a light meter, set at a range pereopods 1-3 are inserted between the gills of 0-200 lux ± 4%, whose sensor was mounted of the same segment and those of the next on the phototube of the microscope. Relative posterior segment (Figs. IB, 2A, 3B-D,F). (percent) transparency was defined as the read­ The epipod of maxilliped 2 is disposed some­ ing of light intensity with the gill inside the what more obliquely in the branchial cham­ field of view divided by the fight intensity ber than the pereopodal epipods (Figs. 2A,B, without the gill X 100 Settings of factors 3E). Its broad proximal portion sits posterior besides magnification (250x.) that affected to the three gills of maxilliped 2 and anterior the intensity of light transmitted through and medial to the anterior arthrobranch of the slide (rheostat, apertures of field and iris diaphragms, condenser) were standardized.

RESULTS Fig. 2. Mmopenaeus similis. Disposition uf yill- Gills and gill-cleaning structures cleauing epipods, exopods. and their setae. Area dis­ played is from box in Figure 1A, shown in Figure IB Removal of the branchiostegite (gill cover) with complete set of gills. A: Branchial chamber, with exposes the gills within the branchial cham­ anterior arthrobranehs of majrilliped 3, pereopods 1-3 removed to reveal inner (medial) layer of gills and posi­ ber (Figs. 1A.B, 3A, Table 1). The podo- tions of gill-cleaning epipods. Anterior arthrobranch of branch of maxilliped 2, the anterior arthro­ maxilliped 2 is hidden medial to the posterior arthro­ branehs of maxilliped 3 and pereopods 1-3, branch of that somite. B: Branchial chamber, all gills the posterior arthrobranch of pereopod 3, removed, showing disposition of multidenticulate setae of gill-cleaning epipods and exopods. aa, anterior arthro­ and the single arthrobranch of pereopod 4 branch; ar, single arthrobranch of pereopod 4; e, gill- form a laterally-positioned outer layer of gills deanir.g epipod; rale, epipod of first maxilliped; m2. max­ (Figs. IB, 3A). Deeper within the branchial illiped 2; m3. maxilliped 3; pa, posterior arthrobranch; chamber, medial to the large anterior arthro­ pd. podobranch; pl-p5. pereopods 1-5 (arrows point to basi-ischial breakage plane, with distal portion of limb branehs of maxilliped 3 and pereopods 1-3, removed); s, scaphognathite (gill bailer); x, pereopodal is an inner layer of gills, composed of the exopod. Unmarked arrows point to the attachment stalk anterior and posterior arthrobranehs of of the anterior arthrobranehs which were removed. Pi P' m3pl

2 mm

^

B

Figure 2. 130 K.T. BAUER maxilliped 3. Its long distal portion contin­ Microscopic observation of an acid-fuchsin- ues medial to both arthrobranchs and lat­ stained mount of this thin-walled structure eral to the pleurobranch of maxilliped 3, indicates that this epipod, like those on the terminating between the latter and the pleu­ pereopods, lacks muscle fibers. Its movement robranch of pereopod 1 (Figs. 2A,B, 3E). The may have been due to its proximity to the epipod of maxilliped 1 is a broad blade (Fig. scaphognathite, whose constant movement 4) situated horizontally ventral to the scaph- draws water into the branchial chamber. ognathite (gill bailer) (Figs. IB, 2A); its outer Epipodal setae with denticulate scales edge lies against the anterior side of the come into contact with almost all areas of podobranch of maxilliped 2 (Figs. IB, 2A, 3A). the gills except for the lateral (outer) sides of The epipods of peropods 1-3 and maxilli­ the outer layer of gills, specifically, the ar- ped 2 are equipped with long, structurally throbranch of pereopod 4, the posterior ar- complex setae (Figs. 2B, 3B.D-F, 5A-C,E,F). throbranch of pereopod 3, and the anterior These setae are located distally on the part arthrobranchs of pereopods 1-3 and maxilli­ of the epipods of pereopods 1-3 that makes ped 3. In looking for possible gill-cleaning contact with the gills (Figs. 2B, 3B-D). The setae associated with these gill areas, it was epipod of maxilliped 2, completely sur­ observed that the exopods of pereopods 1—4 rounded by gills, has these setae throughout (Figs. IB, 2A,B, 3A,C, 6A.B) are bordered by its length (Figs. 2B, 3B,E). Arising from deep long multidenticulate setae, arising from sockets (Fig. 5D), these multidenticulate se­ deep sockets, with similar microstructure tae are naked proximally and studded dis­ (Fig. 6B) as that of the setiferous epipods. tally by an array of digitate scale setules These pereopodal exopods are short, oblong, (Fig. 5E,F). The setae extend among the gills flattened structures (Fig. 7) that have been and gill filaments (Figs. 3F, 5A-C). Due to the observed to rock back and forth, with their location of the epipods, nearly all gills are in long multidenticulate setae sweeping over contact with multidenticulate epipod setae. the lateral surfaces of those gills in the outer Setae of a pereopodal epipod make contact layer (except for the arthrobranch of pere­ with the anterior sides of arthrobranchs of opod 4 and the podobranch of maxilliped 2). the immediately posterior somite and with The setae of these exopods extend up about the posterior sides of the arthrobranchs of as far as the proximal half to two-thirds of the same somite. They extend medial to these the gill (Figs. 2B, 3A,C, 6A). The exopod of a same gills, as well as lateral to the pleuro- given limb sweeps the outer gill(s) of its branchs of the posterior somite. The cover­ samite when moved posteriorly (extended) age given by the longer maxilliped 2 epipod and that of the anterior somite when moved is even more extensive. Its setae make contact anteriorly (flexed). There is a very small with all the gills of its own somite, including (vestigial) exopod on pereopod 5 (Figs. IB, the outer or lateral side of its podobranch, 2A,B, 7) equipped only with short plumose with the anterior side and medial sides of both setae (Figs. 2B, 6C). The exopods of maxilli- arthrobranchs of maxilliped 3, and with the peds 1-3 are long and flagelliform (Figs. pleurobranchs of maxlliped 3 and pereopod 1A,B, 6D, 7), especially those of maxillipeds 1. The epipod of maxilliped 1 lacks the array 2 and 3, and all carry long plumose setae of long complex setae found on other tho­ (Fig. 6D). Neither the exopods of pereopod 5 racic limbs; its outer edge is fringed by short and maxillipeds 1-3 nor their setae come plumose setae (Fig. 3E). However, on the under- into contact with gills (Figs. 1A, 2A,B). sidn of its outer edge, which lies against, the anterior side of the podobranch of maxilliped 2, The scaphognathite and the inner surface there is a scattering of relatively short setae of the branchiostegite, structures observed equipped with denticulate scale setules. to have gill-cleaning setae in some other decapods, were examined. No long multiden­ Any movement of an appendage that car­ ticulate setae were observed extending over ries a setiferous epipod causes the epipod the gills from the posterior edge of the scaph­ and its setae to be moved among and over ognathite; instead, only short plumose setae the gill filaments. Multidenticulate setae occur there (Fig. 3E). The inner surface of thus are scraped over and jostled among the the branchiostegite is largely devoid of setae gill filaments. Observations on living shrimps with the exception of a group located posteri­ showed that the epipods of pereopods 1-3 do orly (Fig. 8A). Setae from this group arise not move spontaneously, only with move­ from an area of the branchiostegite that lies ment of the limb to which they are attached. directly against the arthrobranch of pere­ The epipod of maxilliped 2 was observed to opod 4, a gill which has no contact with beat or rock within the gill chamber when either epipodal or exopodal setae. In the the second maxilliped was not in motion. dorsal part of the sctal patch, the setae (Fig. GILL-CLEANING MECHANISMS OF K. S1MIL1S 131

Fig. 3. Rimapenaeus similis. Branchial chamber and of gill chamber (upper right in B), with anterior arthro- epipods. A: Branchial chamber with gill cover(branchio- branch of maxilliped 3 and podobrancli of maxilliped 2 stegite) removed to reveal gills, basal portion of epipods. removed to reveal proximal portion of setiferous epipod and exopods (cf. Fig. IA.B); dorsal at top. B: Anterior of maxilliped 2, with distal part extending under (me­ end of branchial chamber, anterior arthrobranchs and dial to) posterior arthrobranch of maxilliped 3; dorsal at maxillped 2 podobrancli removed to reveal posterior top. F: Setiferous distal branch (arrowl of epipod of arlhrnbranclis, other underlying gills, and epipods of pereopod 1 extending between posterior arthrobranchs maxilliped 2 and pereopods 1 and 2 (cf. Fig. 2A), exopods of pereopods 1 and 2 (anterior arthrobranchs removed, also removed for clarity; dorsal at top. C: Arrangement dorsal to left), aa, anterior arthrobranch; c, gill-cleaning of exopods, epipods, and gills on pereopods 1-3 upon epipod; mle, epipod of first maxilliped; m2, maxilliped 2; removal of gill cover; dorsal at top. D: Blade of epipod of m3, maxilliped 3; pa, posterior arthrobranch; pi—p3, pereopod 1 extending under and between gills of pereo­ pereopods 1-3; s, scaphognathite (gill bailer); x, pereopo- pods 1 and 2, stem of epipod of pereopod 2 arising from dal exopod. Scale bar — 1.8 mm in A, 1.0 mm in B, 800 the latter's coxal segment; dorsal to left. E: Anterior end run in C, 440 um in D, 600 pm in E, and 540 |im in F.

8B,C), arising from deep sockets, are densely with digitate scale setules only, similar to covered with long slender setules proximally those described above on pereopodal exo­ (Fig. 8B,C) but distally with digitate scale pods and epipods. There is a sparse scatter­ setules (Fig 8C,F)) From dorsal to ventral ing of very short hut. typical multidenticu­ in this setal group, the setae change in type late setae more anteriorly on the inner side from plumodenticulate to multidenticulate, of the branchiostegite (Fig. 8A). 132 R.T. BAUER particles could be observed being dislodged from experimental gills but not control gills, which were little fouled. Examination of equivalent control and experimental gills by scanning electron microscopy revealed the heavier fouling on experimental gills com­ pared to little or none on controls (Fig. 9A- F), with fouling particles trapped among branches of gill filaments (Fig. 9C.E). Bacte­ rial or other epibiotic fouling was not notice­ able on either experimental or control gills, except perhaps for that associated with foul­ ing aggregates on experimental gills. Particulate fouling was quantitatively compared between control and experimental gills by measuring their degree of transpar­ Fig. 4. Rimapenaeus similis. Relative size and shape ency to light, which is lowered by fouling. of thoracic epipods. nil,3, epipods of maxillipeds 1,3; Medians of percent transparency for experi­ pl-p3. epipods of pereopods 1—3. mental gills were always lower than those of control gills, and there was only slight over­ lap in 95% confidence limits on medians for Observation of living shrimps for possible the anterior arthrobranchs (Fig. 10). The gill-brushing behavior null hypothesis of no difference between ex- perimentals and controls was tested for each The activities of 40 Rimapenaeus similis pair of gills with the Wilcoxon matched- individuals were observed for a total of 19 hr pairs signed-ranks test and was rejected in during the daytime and 90 hr at night, when all cases (P < 0.002). this species is most active. Antennular and antennal grooming behaviors with the third In the exopod-ablation experiment, par­ maxillipeds and first pereopods were ob­ ticulate fouling could be readily observed on served, as well as infrequent bouts of pick­ the lateral (outer) surfaces of the anterior ing at various parts of the body with the arthrobranchs of the outer gills (maxilliped chelipeds (pereopods 1-3). There was never 3 and pereopods 1-3) in the experimental any insertion of the chelipeds into the gill but not in the control gill chambers. When chamber nor any other sign of gill brushing gills were mounted in order to measure light by these appendages. transparency, it was observed that there was usually a gradient of fouling in the experi- Experimental testing of gill-cleaning function mentals, with more fouling proximally. How­ ever, the distal end of the gill also had notice­ In the epipod-ablation experiment, epi­ able fouling in the experimental gills but not pods were removed from the branchial cham­ in controls (Fig. 11). As in the epipod-abla­ ber of one side (experimental) but not the tion experiment, fouling was particulate, other (control) before treated shrimps were with little observation of epibiotic fouling on exposed to fouling on a recirculating water control or experimental gill filaments. Medi­ table. Treated shrimps preserved after the ans of relative gill transparency were lower experiment were first examined by removal in experimental gills than in controls, and of the gill cover and observation through the there was overlap in 95% confidence limits dissecting microscope. There was no visible only for the anterior arthrobranchs of the sign of fouling on the lateral surfaces of the third maxillipeds (Fig. 12). However, the outer gills (anterior arthrobranchs) when null hypothesis of no difference in relative the branchiostegite was removed in either transparency between control and experimen­ the experimental or control chambers. How­ tal pairs of gills was rejected in all cases (Wil­ ever, when the outer gills were removed coxon test, P s 0.004 in all comparisons). from the experimental gill chamber, particu­ late fouling could be readily observed on the inner gills that lie medial to them, as well as DISCUSSION on their own anterior and posterior edges, and especially on their medial sides. Qualita­ The gill-cleaning system of Rimapenaeus tively, particulate fouling on gills from the similis is composed of a combination of struc­ control gill chamber was difficult to observe. tures that carry multidenticulate setae. The When gills were mounted for quantitative primary mechanism consists of the setifer- measurements (see below), less adherent ous epipods of maxilliped 2 and pereopods 1—3. Their long multidenticulate setae are GIl.I.-Cl.F.AXIXG MECHANISMS OF K. SIM/US 133

Fig. 5. liima/ienat'ussimilis. Multidenticulate epipo- epipodal seta (arrows) among gill filaments. D: Base and dal .setae. A: Setae at distal end (arrow) of pereopodal deep socket of epipodal seta. E: Portions of two epipodal epipod in contact with adjacent posterior arthrobranchs setae; note digitate scale setules on setal shafts. (top, bottom) as well as to a pleurobranch (between and F: Digitate scale setules on shaft of epipodal seta. Scale medial to the arthrobranchs) Idorsal to left). B: Epipodal bar = 230 pm in A, 80 pm in B. 20 pm in C, 5 pm in D, 4 setae (arrows) in contact with gill filaments. C: Single pm in E, and 3 pm in F. covered with digitate scale setules very simi­ the inhalant water from settling or remain­ lar in microstructure to setae that have been ing on respiratory surfaces. This gill-clean­ shown experimentally to clean the gills in a ing function of epipods is confirmed by the variety of decapods, e.g., cheliped brushes fouling of gills that occurs when epipods are of caridean shrimps (Bauer, '79) and seto- removed. In the crayfish Procambarus clar- branch setae in crayfish (Bauer, '98). In R. kii, Bauer ('98) showed that setobranch se­ similis, when the epipods are moved pas­ tae, arising directly from the limb coxa, sively or indirectly by feeding or locomotory cleaned a comparable inner layer of gills. movements of the limbs that bear them, Although epipodal setae clean most gill their setae are jostled among and over the areas in Rimapenaeus similis, they do not gill filaments with which they make contact, reach to the lateral surfaces of the outer preventing particulate matter carried in by layer of gills, i.e., the anterior arthrobranchs 134 R.T. BAUER

Fig. 6. Rimapenaeus similis. Rxopods and setae. A: vestigial exopod of pereopod 5. D: Flagelliform exopod of Pereopodal exopods equipped with multidenticulate se­ third maxilliped with plumose setae, e, epipod; p2, coxa tae extending over basal part of outer gills (anterior of pereopod 2; x, pereopodal exopod. Scale bar - 800 uni arlhrobranchs). B: Portion of multidenticulate exopodal in A, 4 pm in B, 70 pm in C, and 220 pm in D. seta with digitate scale setules. C: Plumose setae on of maxilliped 3 and pereopods 1-3, the poste­ experimentally removed, but sediment and rior arthrobranch of pereopod 3, and the particles did accumulate when the exopods single arthrobranch of pereopod 4. Except (but not epipods) were removed. The primi­ for pereopod 4 arthrobranch, the lateral sur­ tive, flagelliform-type of exopod equipped faces of these gills are swept by multidenticu­ with plumose setae, with a natatory or cur­ late setae on specialized exopods of pereo­ rent-producing function, is retained on pods 1-4. Fouling of these areas did not maxillipeds 2-3. In pereopods 1-4, however, occur when epipods (but not exopods) were the exopod has been modified into a shorter, flattened form, equipped with long, multiden­ ticulate cleaning setae that sweep back and forth over the gills. The motion of these exopods is a retention of ancestral natatory movements used instead for gill cleaning. 0.5 mm Interestingly, the very small exopod of pere­ opod 5, vestigial as far as size and any obvi­ ous function are concerned, retains the plu­ mose setae of a primitive, natatory exopod. In the crayfish Procambarus clarkii, which lacks pereopodal exopods, Bauer ('98) showed that gill surfaces directly medial to the bran- snmnciDiir^ chiostegite (gill cover) were cleaned by a dense 1.0 mm field of multidenticulate setae arising from the inner side of the branchiostegite itself. In Rimapenaeus similis, the branchiostegite Fig. 7. Rimapenaeus similis. Relative size and shape of exopods. m.'i, Hagelliform exopod of third maxilliped; is largely devoid of such cleaning setae (cf. pl-p5, exopods of pereopods 1—5. Fig. 8Awith Fig. 5B in Bauer, '98), and it is the (III.^CLEANING MECHANISMS OF H. SI.Ml US 135

Fig. 8. Rimapenaeus similis. Cleaning setae of the A. C: Distal (above) and proximal (below) portions of branchiostegite (gill cover). A: Inner side of right, bran­ setae located in patch indicated by arrow in A. D: Multi- chiostegite, showing patch of setae (arrow, upper right) denticulate scale setules on distal ends of setae from the in contact with the arthrobranch of pereopod 4. B: Higher inner side of the branchiostegite. Scale bar = 1.4 mm in magnification of plumodenticulate setae indicated in A, 300 pm in B, 20 pm in C, and 2 pm in D. exopods of pereopods 1-4 that clean the gill of the scaphognathite above it, would cause surfaces just medial to the branchiostegite. these setae to clean the gill filaments below. A few specialized setal groups appear to The scaphognathite sweeps long multiden­ clean some limited, very specific gill areas. ticulate setae over the lateral surface of gills The only gill that is not in contact with exopo- in some decapods, e.g., a few caridean dal and epipodal setae, the single arthro­ shrimps (Bauer, '79; Suzuki and McLay, '98) branch of pereopod 4, appears to be cleaned and a thalassinid "mud lobster" (Batang and by the sole field of setae on the inside of the Suzuki, '99), and these scaphognathite setae branchiostegite. These setae, located di­ are a probable passive cleaning mechanism. rectly opposite the pereopod 4 arthrobranch, No such setae were observed on the scaphog­ bear digitate scale setules distally, protrude nathite in Rimapenaeus similis, in which into the gill, and presumably jostle among the long exopodal cleaning setae sweep the its gill filaments during body movements. lateral surface of the outer layer of the gills. Although the other thoracic epipods are In the crayfish Procambarus clarkii, which gill-cleaning structures, the epipod of the also lack scaphognathite setae, another pas­ first maxilliped primarily serves not in clean­ sive mechanism, multidenticulate cleaning ing but rather as part of an anterodorsal setae on the inside of the gill cover, cleans funnel through which respiratory water en­ these gill surfaces, as discussed above. In R. ters the gill chamber in penaeids (Young, similis, these are only developed over the '59; Dall et al., '90). It is not equipped with most posterior gill, which is not reached by long setae as are the other thoracic epipods; either exopodal nor epipodal cleaning setae. however, where it lies against the anterior Active brushing of the gills by chelipeds is side of the podobranch of its somite, there is the gill-cleaning mechanism of some carid­ a minor field of typical multidenticulate clean ean groups, anomuran crabs, and callianas- ing setae. Presumably, movements of the epi­ sids. Chelipeds are inserted among the gills pod, either inherent or caused by the beating and brushed vigorously among them. The R.T. BAUER

Fig. 9. Rimapenaeus simiiis. Results of the epipod- gill (compare with D) shown in A. D: Lack of particulate ablation experiment: comparison of fouling from experi­ fouling among filaments from control gill (compare with mental and control gills of the same specimen. A: Medial C) shown in B. E: Higher magnification of particulate side of anterior arthrobranch (distal to left) of third fouling on experimental gill (compare with F) from A maxilliped from experimental branchial chamber. Box and C. F: Higher magnification of clean filaments from shows region of gill magnified in C and E. B: Same control gill (compare with E) shown in B and D. Scale gill type and view as A, but from control branchial bar = 360 pm in A and B, 60 pm in C and D, and 25 pm chamber. Box shows region of gill magnified in D and F. in E and F. C: Particulate fouling among filaments of experimental animal can selectively pick off material us­ where such fouling may interfere with gas ing fingers of the chelae, and it can concen­ exchange, ion exchange, and excretion. In trate its efforts on one area over another. this study, significant particulate fouling oc­ Not surprisingly, cheliped cleaning of gills curred on gills when the passive cleaning has been shown experimentally to be quite mechanisms (exopods, epipods) were re­ efficient at preventing both particulate and moved. Similarly, in the crayfish Procamba- epibiotic fouling on the gills (Bauer, '79; rus clarkii, massive particulate fouling oc­ Pohle, '89). Passive gill-cleaning mecha­ curred in the experimental gill chamber nisms are certainly effective in preventing when the setobranchs, the primary gill- the accumulation of sediment and detrital cleaning structures, were removed from cray­ particles from accumulating on gill surfaces, fish exposed to fouling in commercial ponds GILL-CLEANING MECHANISMS OF ft. SIMILIS 137 Epipod Ablation Experiment cleaning system to that of the crayfish Pro- cambariis clarkii suggests the hypothesis, still untested, that the penaeid gill-cleaning 65 system is not effective against epibiotic foul­ ing. It is further hypothesized that, while the gills are kept free of particulate fouling by 60 the combined action of the setiferous epipods and pereopodal exopods, molting is the only &55 escape ol'R. similis from epibiotic fouling. 2 m 50 The evidence that passive gill-cleaning o. mechanisms are primitive and that cheliped n brushing of gills is derived in the Decapoda § 45 has been given in Bauer ('81, '89, '98). When cheliped brushing evolves in a group, pas­ * 40 sive gill-cleaning mechanisms become super­ - fluous and maintenance of them is selected 35 Maa against. Since there are various forms of Mpa passive gill cleaning, the question arises: 30 which was or were the mechanisms used by the shrimp-like decapod ancestor (descrihed in Burkenroad, '81)? For the primary gill- Gill Type and Treatment cleaning mechanism, the decapod ancestor may have had both setiferous epipods and Fig. 10. Rimapenaeus similis. Results of epipod- setobranchs. Borradaile ('07) considered seto­ ablation experiment: comparison of particulate fouling on gills from control and experimental branchial cham­ branch papillae to be derivatives of the epi­ bers, using measures of gill transparency to transmitted pods, and Batang and Suzuki C99) demon­ light. Medians and 9B% confidence limits of percent strate the presence of both setiferous epipods transparency arc given for the gills of the third maxilli- and setobranch setae in the passive gill- ped and third percopod from the control (epipods pre­ sent) and experimental (epipods removed) branchial cleaning system of the mud lobster Thalas- chambers (n = 25 individuals). Maa, Mpa, and Mpl, ante­ sina anomala. Setiferous epipods or seto­ rior arthrobranchs, posterior arthrobranchs. and pleuro- branchs are the primary mechanisms in branchs. respectively, of third maxillipeds; Paa, Ppa, and groups in which cheliped brushing has not Ppl, anterior arthrobranchs, posterior arthrobranchs. evolved (Bauer, '81, '89, '98). However, in any and pleurobranchs, respectively, of third pereopods. decapod group in which passive gill cleaning occurs, these primary mechanisms do not clean the gill filaments on the lateral sur­ and a natural swamp environment (Bauer, faces of the gills just medial to the gill cover. '98). However, the presence of setobranchs Since the ancestral decapod, a swimming in crayfishes did not prevent heavy fouhng shrimp-like organism, must have had well- on control gills by bacteria and a sessile proto­ developed pereopodal exopods equipped with zoan, Cothurnia variabilis. This lack of effi­ plumose (natatory) setae, cleaning of the ciency in preventing epibiotic fouling was attrib­ outer filaments must have been performed uted to the nonselective movements of the by some non-exopodal mechanism, e.g., bran- gill-cleaning setobranch setae (Bauer, '98). chiostegal setae or scaphognathite cleaning In the present study treated individuals setae. In most decapods, pereopodal exopods oi'Riinupenueus similis were exposed to foul­ are absent or reduced and no longer nata­ ing in a recirculating water system, an envi­ tory. In Rimapenaeus similis and perhaps other ronment that can be conducive to heavy penaeoid shrimps, the reduced exopods have epibiotic fouling (Lightner, '831. However, been modified into gill-cleaning structures. the shrimps were maintained individually The absence, presence, and relative impor­ at low density, excess food and wastes were tance of gill-cleaning setae on reduced exo­ removed daily, and, because of an effective pods, on the inner side of the gill cover, and filter system, water quality was high. No fringing the scaphognathite will have to be measurable epibiotic fouling was observed described in more decapod groups in order to on control or experimental gills of treated hypothesize primitive and derived states in shrimps, suggesting that epibiotic fouling these characters among the Decapoda. pressures were low in the system used and that the ability of the gill-cleaning system of The pereopodal exopods were shown in R. similis to resist epibiotic fouling was not this study to be important in cleaning the tested in these experiments. However, the gill surfaces just medial to the gill cover. It is similar mode of operation of this passive gill- not known if the exopods of other penaeoid R.T. BAUER

Fig. 11. Rimapenaeus similis. Results of the exopod- ing (compare with Al. Box shows area magnified in D. ablation experiment: compar.son of fouling from experi­ C: Particulate fouling on filaments in experimental gill mental and control gills of the same specimen. A: Lat­ (compare with Dl from boxed area in A. D: Lack of eral side of distal end of anterior arthrobranch, pereopod fouling of filaments of control gill (compare with C> from 2, from experimental branchial chamber, showing par­ boxed area in R R,F- High magnification nf particulate ticulate fouling (compare with B). Box shows area mag­ fouling on experimental gill shown in A. Scale bar - 140 nified in C. B: Same gill type and view as A, but from pm in A and B, 55 pm in C and D, 9 pm in E, and 7 pm control branchial chamber. Note relative absence of foul­ in P. genera participate in gill cleaning. Exopods Aristeidae and Benthesicymidae lack pere- similar to or smaller in size than those of opodal exopods. Exopods of representative Rimapenaeus similis are present on all penaeoid species need to be studied to deter­ pereopods in genera of Penaeidae (except mine if these structures are involved in gill Arlemesia; Perez Farfante and Kensley, '97) cleaning as in R. similis. When they are and Solenoceridae. Preliminary observa­ absent or do not function in gill cleaning, the tions in Litopenaeus setiferus show that the mechanisms that may replace them, such as setae on its exopods are multidenticulate branchiostegal, scaphognathite, or other like those of R. similis; however, those of cleaning setae, need to be determined. An Solenocera vioscai are not. All species of array of such characters from penaeoid gen- GILL-CLEANING MECHANISMS OF R. SIMIUS 139 salambria, and all species of Trachypenaeus Exopod Ablation Experiment (s.s.), the number of pereopodal epipods may be reduced to only one (pereopod 3), with only it and that of maxilliped 2 available for 60 gill-cleaning. Studies are needed to deter­ mine if and how that deficiency of basic gill cleaning equipment is compensated for in 55 these taxa. fc c 50 ACKNOWLEDGMENTS 9>t_ TO I thank Dr. Frank Truesdale for his partici­ Q. 45 pation, help, and good company during trawl­ to ing cruises in which shrimps were collected (c0 for study. I thank Dr. Isabel Perez Farfante «— r- 40 for reading and making helpful comments ^ on the manuscript. This is Contribution No. 69 of the Laboratory for Crustacean Research. 35 LITERATURE CITED Bntang Z, Suzuki H. 1999. Gill-cleaning mechanisms of 30 the mud lobster Thalassina anomuln Herbsl. 1804 (Decapoda: Thalassinida: Thalassinidae). J Crusta­ cean Biol 19(4):in press. Gill Type and Treatment Bauer RT. 1979. Antifouling adaptations of marine shrimp (Decapoda: Caridea): gill cleaning mecha­ Fig. 12. Rimapenaeus srmilis. Results of the exopod- nisms and cleaning of brooded embryos. Zool .1 Linn ablation experiment: comparison of particulate fouling Soc-65:281-303. on gills from control and experimental branchial cham­ Bauer RT. 1981. Grooming behavior and morphology in bers, using measures of gill transparency to transmitted the decapod Crustacea. J Crustacean Biol 1:153-173. light. Medians and 95% confidence limits are given for Bauer RT. 1989. Decapod crustacean grooming: func­ percent transparency of the anterior arthrobranchs of tional morphology and functional signilcance. In: Fel- the third maxilliped and pereopods 1-3 from the control genhauer BE, Watling L, Thistle AB, editors. Func­ (exopods present) and experimental (esopods removed) tional morphology of feeding and grooming in branchial chambers (n - 25 individuals). M3, P1-P3, Crustacea. Rotterdam: AA Balkema. p 49-73. anterior arthrobranchs of the third maxillipeds and Bauer RT. 1998. Gill-cleaning structures of the crayfish pereopods 1-3. respectively, Procambarus clarkii (Astacidea: Cambaridae): experi­ mental testing of setobranch function. Invert Biol 117:29-143. Borradaile LA. 1907. On the classification of the decapod era will be extremely useful in studies on the crustaceans. Ann Mag Nat Hist Ijondon 7:407—486. evolution of gill cleaning, as well as in analy­ Burkenroad MD. 1981. The higher taxonomy and evolu­ tion of Decapoda (Crustacea). Trans San Diego Soc ses of phylogenetic relationships in the Nat Hist 19:251-268. Penaeoidea and other decapod crustaceans. Dall W. 1957. A revision of the Australian species of In penaeoid shrimp, the primary gill- Penaeinae. Aust.J Mar Freshwater Res 15:136-230. cleaning mechanism, setiferous epipods, is Dall W. Hill BC, Rothlisberg PC, Sharpies DJ. 1990. The often reduced. In the families Aristeidae, biology of the Penaeidae. Adv Mar Biol 27; 1-489. Lightner DV. 1983. Diseases of cultured penaeid shrimp. Benthesicymidae, and Solenoceridae, there In: McVey JP, editor. CRC handbook of mariculture. are epipods on every thoracic limb back to vol I. Crustacean aquaculture. Boca Raton. FL: CRC and including the fourth pereopod (bran­ Press, p 289-320. chial formulas given in Perez Farfante and Perez Farfante I. 1971. A key to the American Pacific Kensley, '97). In Rimapenaeus, as well as in shrimps of the genus Trachypenaeus (Decapoda, Penaeidae) with the description of a new species. Fish several other genera in the Penaeidae and Bull 69:635-646. all Sicyoniidae, the epipod of the third maxil­ Perez Farfante I, Kensley B. 1997. Penaeoid and serges- liped is absent. In R. similis, it was observed toid shrimps and prawns of the world. Mem Mus Nat that the epipod of maxilliped 2, because of Hist Nat 175:1-233. its size, greater array of multidenticulate Pohle G. 1989. Gill and embryo grooming in lithodid cral)s: comparative functional morphology based on setae relative to the pereopodal epipods, and Lithodes maja. In: Felgenhauer BE, Watling L, Thistle its oblique positioning in the branchial cham­ AB. editors. Functional morphology of feeding and groom­ ber, was able to take the place of the "miss­ ing in Crustacea. Rotterdam: AA Balkema. p 75-94. ing" epipod of the third maxilliped. This may Suzuki H, McLay CL. 1998. Gill-cleaning mechanisms of also be the case in other penaeoid species Paratya curvirostris (Caridea: Atyidae) and compari­ sons with seven species of Japanese aytid sin imps. J with the same pattern of epipod number. In Crustacean Biol 18:253-270. Parapenaeun, Parapenaeopsis, and espe­ Youiifc' JH 1959. Morphology of the white sliri mp Penaeus cially in some species of Me.gokris, Trachy- setiferus (Linnaeus 1758). US Fish Wild Serv Fish Bull 59:1-168.