The effects of residential tubes on reproductive behaviors in Microdeutopus gryllotalpa (Costa) (Crustacea: Amphipoda)

J. Exp. Mar. Biol. Ecol., 1989, Vol. 128, pp. 117-125 Elsevier

JEMBE

117

01252

The effects of residential tubes on reproductive in Microdeutopus gryllotalpa (Costa) (Crustacea:Amphipoda)

behaviors

Betty Borowsky Osborn Laboratories of Marine Sciences, New York Aquarium, Brooklyn, New York, U.S.A. (Received

28 October

1988; revision

received

13 February

1989; accepted

6 March

1989)

Residential tubes of receptive females of the amphipod crustacean Mcrodeutopus gryllotulpa (Costa) stimulate pair-formation behavior by the male. This suggested that females might mark their tubes in some way, a phenomenon previously undescribed in Crustacea. However, experimental data, presented here, disproved this hypothesis. Therefore, an alternative hypothesis was proposed: tubes enhance individual survivorship, so it is advantageous for animals to conduct as many activities inside them as possible. Since M. gryllotalpa may be found in the intertidal zone, even though individuals die within minutes of exposure to the air, the possibility that the tubes protect occupants against exposure was tested. Experiments indicated that residence within tubes can protect occupants from exposure. Thus, the results of this study support the hypothesis that tubes stimulate reproductive behaviors because they enhance survivorship and thus enhance reproductive potential.

Abstract:

Microdeutopus gryllotalpa; Reproduction;

Key words: Amphipod;

Residential

tube

INTRODUCTION

Crustacea are an extremely diverse group and it is not surprising that the stimuli for their reproductive behaviors are also varied (Salmon, 1983, Table I). Some species employ visual displays (members of the fiddler crab genus Uca; Crane, 1975); some species employ pheromones, either waterborne (crabs Portunus sanguinolentus, Carcinus maenas, and Callinectes sapidus; Eales, 1974; Christofferson, 1978; Gleeson, 1980, respectively), or contact pheromones (the amphipod Gammaruspalustnk; Borowsky & Borowsky, 1987). Previous observations revealed that the residential tube of receptive females of the amphipod Microdeutopus gryZZot&a (Costa) was required for the males’ expression of pair-formation behavior (Borowsky, 1983a). This led to the hypothesis that females mark their tubes, a phenomenon previously undocumented for Crustacea. The first objective of the work described here was to test this hypothesis by examining the effects of the tubes of other conspecifics on male behaviors. Correspondence address: B. Borowsky, Osborn Laboratories Boardwalk West 8th Street, Brooklyn, NY 11224, U.S.A. 0022-0981/89/$03.50

0

1989 Elsevier

Science

Publishers

of Marine

B.V. (Biomedical

Sciences,

New York Aquarium,

Division)

B. BOROWSKY

118

All individu~s of ~icrude~topus g~llota~a (Costa) construct tubes by gluing debris together with sticky threads that are secreted through pores in the anterior pereiopods. When submerged, the tubes are open at both ends and are one to three times the length of the constructor (S-IS mm long). The tubes are somewhat elastic, so animals can move about freely inside, and the tubes can accommodate a heterogametic pair (tube-sharing is a regular feature of M. gryllutalpa’s pattern of reproductive behavior (Borowsky, 1983a). Since the tubes are constantly reworked by the resident animal, which requires continuous secretions, it was hypothesized that the threads might contain different chemicals indicating the female’s reproductive condition. The bioassay used to score male-reproductive behaviors was “intermittent pleopod beats” (IPBs). In M. gryllotalpa, courtship entails both tube-sharing and IPBs (Borowsky, 1983a). The latter is a distinct easily recognized behavior which males generally express to receptive females. It involves the strong rapid beating of the pleopods (ventral abdomin~ limbs which generate a respiratory cu~ent) for = 1 S, followed by a rest of l-2 S. The sequence is usually repeated a few times. While males might enter a tube for either protection and/or reproduction, IPBs are only expressed in a reproductive context. Therefore, IPBs alone were scored in the experiments reported here. The first group of experiments tested and disproved the hypothesis that females chemically mark their tubes. Therefore, a second simpler hypothesis was proposed: male-reproductive behaviors are only expressed in the presence of tubes because the tubes are necessary for the animals’ survival. One obvious way that the tubes might enhance survival would be to protect the animals against desiccation. Although this species is found in the intertidal zone, animals exposed to the air die within minutes. Therefore, the ability of the tube to protect animals against exposure was investigated.

MATERIALS

I.

EXPERIMENTS

TO TEST

EFFECTS

AND

OF TUBES

METHODS

ON MALE-REPRODUCTIVE

BEHAVIORS

The nature of the specific aspects of the female’s tube which stimulate reproductive behaviors in males was examined. If a chemical stimulus is involved, then contact with the tube should elicit the males’ reproductive behaviors whether the male is inside or outside the tube (Experimental Series A). Further, if a chemical stimulus is involved, then the tubes of receptive females should elicit a greater frequency of male behaviors than any other type of tube, either animal (Series B) or artificial (Series C, D). Test females were taken from laboratory cultures and isolated in individual IO.%cm diameter glass-culture dishes. They were maintained at 20 “C (15 : 9 = L : D) in seawater of 29-30x, and provided with thawed Ulva ~act~ca thalli ad libitum for food and tube-balding materials. Each female was observed daily until it molted and then used for the experiments either 2 or 6 days later. 2-day postmolt females generally do not elicit

REPRODUCTIVE

BEHAVIORS

IN MICRODEUTOPUS

GRYLLCJTALPA

119

male-reproductive behaviors (are nonreceptive) and &day postmolt females are generally receptive (Borowsky, 1983a). Males were also isolated from culture and were kept from females for 7 days before testing. Each test was begun by pipetting a male directly onto a tube cont~ning a female, alone in a dish of seawater. Observations were made with a dissecting microscope and the occurrence of IPBs was noted. All animals were tested only once, then discarded. Series A These experiments were performed to determine whether reproductive behaviors would be expressed outside the tubes of receptive females. Each of 40 females was evicted from its tube by gently prodding with a needle. 20 females were permitted to reenter their tubes but the other 20 females were prevented from reentering by closing off the ends of the tubes with flat glass weights. The behavior of the males who encountered females inside their tubes was compared with the behaviors of those who encountered the females forced to remain outside. Series 3 These experiments were designed to determine whether any tube, or only tubes constructed by receptive females, could stimulate Moe-reproductive behavior (IPBs). Five experiments, of 20 replicates each, were conducted in: (1) receptive females’ own tubes; (2) other receptive females’ tubes; (3) nonreceptive females’ tubes; (4) males’ tubes; and (5) Amp&hoe valida females’ tubes. [A. valzila was chosen because it is sympatric with M. gqdlotalpa, adapts well to laboratory conditions, and exhibits a pattern of reproduction similar to A4.gryilotalpa (Borowsky, 1983b)]. First, the original occupants of the tubes were evicted from their tubes and removed from the dish. Then, one receptive female was introduced into each dish. The females entered the tubes within seconds; then, a male was introduced into each dish. The occurrence of IPBs was recorded. Series C These experiments were designed to ascertain if IPBs would be expressed to receptive females in nonanimal tubes. Capillary tubes (id 1.6-1.8 mm) were chosen to be the artificial tubes because they are about the same width as M. gryllotalpas’ tubes and they are transparent, permitting direct observations of behavior under the microscope. Sections about twice the body length of each female (zz 10 mm) were employed. A slightly different procedure had to be followed in these as opposed to the previous tests because females would not enter tubes when both the female and the capillary tube were placed directly in seawater. However, females readily entered capillary tubes if they were placed on the hand and brought close to the opening of a capillary tube filled with seawater. Two experiments, consisting of 20 replicates each, were performed. The first experi-

B. BOROWSKY

120

ment consisted of placing females inside the capillary tube into a culture dish containing seawater only, then introducing a male and observing the occurrence of IPBs, The second experiment was identical to the first, except that the capillary tube containing the female was placed inside an empty M. gryllotulpa male’s tube. Series

D

Obse~ations of the expression of IPBs to females under a piece of Ulva were made to determine whether a tube or merely a hiding place is a prerequisite for the expression of IPBs. 20 receptive females were placed in individual dishes with a piece of Ulva. Then, a male was introduced into each dish and the occurrence of IPBs was observed. II.

EFFECTIVENESS

OF TUBE

IN PROTECTING

AGAINST

EXPOSURE

Test females and males were obtained and treated before testing as described in Expt. I. Two measures were taken to test the protection afforded to tube occupants; mortality and the length of the intermolt period. Only females were employed in these studies because the time of their molts is more predictable than males’. Females molt about every 7 days at 20 “C (Borowsky, 1980a). In contrast, the times of the males’ molts are variable and relatively infrequent. On the 5th day after its molt, each of 120 females was placed in a clean glass culture dish with fresh seawater with a thin layer of beach sand. Then, each female was assigned to one of the following three treatment groups (40 females * group - ‘): (1) water changed daily; (2) females exposed for 1 day (l-day exposure group). The water was decanted gradually from the dish on the 6th day after the female’s previous molt, leaving the sand moist. Fresh seawater was introduced on the 7th day, then changed once daily thereafter; (3) females exposed for 2 days (2-day exposure group). Treated as the l-day exposure group except seawater introduced at the end of the 8th day. To investigate how the tubes might be protecting the females against exposure in more detail, 39 females were observed in their tubes under a dissecting microscope during their exposure days. Each female in each group was observed daily until it either molted or died. Intermolt periods were calculated using the days on which the casts were observed in the dishes. RESULTS

I. EFFECTS

OF TUBES

ON EXPRESSION

OF IPBS

Series A Significantly fewer males expressed IPBs when they encountered females on top of their tubes than when they encountered females inside them (1 of 20 vs. 17 of 20 times, respectively; Fisher’s exact probability test, P = 2.0 - ‘). Thus, cooccupancy of the tube, not just contact with it, is important in eliciting IPBs.

REPRODUCTIVE

BEHAVIORS IN MICRODEUTOPUS

GRYLLOTALPA

121

Series B

There was no significant difference in the frequency of IPBs expressed to receptive females in the live different types of tubes (Table I; xz = 3.770, P > 0.05). Thus, receptive females’ tubes did not enhance and A. valida’s tubes did not diminish the males’ reproductive behaviors. TABLE I

Males’ expression of IPBs to receptive female M. gryllotalpain different types of tubes. Expression of IPBs

Type of tube Receptive females’ own Other receptive females’ Nonreceptive females’ Males’ A. valida

Yes

No

16

4

11

3

13

I

13

I

13

1

females’

Series C

In the 20 tests involving females in capillary tubes, seven females left the capillary tubes immediately upon their introduction into the dish and 12 males did not attempt to enter the capillary tubes or investigate their contents. Thus, only one male expressed IPBs under these conditions. In addition, no IPBs were expressed when females and males were placed in dishes with a fresh piece of Ulva. Females swam rapidly around the dish, and did not remain quiescent under the Ulva, which prevented encounters. In contrast, females remained in the capillary tubes when they were placed inside males’ tubes, and 10 of 20 males expressed IPBs under these conditions. This was significantly greater than when capillary tubes were not placed in males’ tubes [ 1 of 40 (Ulva and capillary tubes combined) vs. 10 of 20; Fisher’s exact probability test; P = 2.5- 5]. Thus, cooccupancy inside an animal tube seems to be a prerequisite for the expression of IPBs but the type of animal that builds the tube is relatively unimportant. II.

EFFECTIVENESS

OF TUBE

IN PROTECTING

AGAINST

EXPOSURE

Mortalities

Most females survived to the molt regardless of how long they were exposed. However, there was a significant difference in mortalities among the three groups (Table II: xz = 5.099, P < 0.05, one-tailed). A greater proportion of females died when they were exposed to the air for 2 days than when they were not exposed or when they were

122

B. BOROWSKY

exposed for only 1 day (Table II: considering only females that molted after observations were begun; 24 females survived to the molt and 14 died in the 2-day exposed group; 57 females survived to the molt and 12 died in the 0- and l-day exposed groups combined: x: = 5.112, P-C 0.05, one-tailed. In contrast, there was no difference between the 0- vs. the l- and 2-day exposed groups combined; XT = 0.394, P < 0.05). TABLE II Frequency

of deaths

and molts of females

never exposed,

exposed

Exposure

Molt Die Molt before observations begin Total Intermolt periods

for 1 day, or exposed

for 2 days.

(days)

0

1

2

Total

26 6 8

31 6 3

24 14 2

81 26 13

40 6.5 f 0.6 SE

40 6.5 f 0.7 SE

40 7.1 k0.7SE

120

Intermolt period lengths

Exposed females molted significantly later than unexposed Kruskal-Wallis one-way ANOVA, H, = 8.9, P < 0.05).

females (Table II:

Frequency of molts during periods of exposure

On average, females molted about as often during periods of exposure as during periods of submergence on Day 6 and on Day 7 past their previous molts (see Table III). Thus, merely removing water from the dish did not inhibit molting. TABLE III Frequency

of female molts in tubes covered

Day 6 Tubes exposed Tubes covered Totals

Day 7* Tubes exposed Tubes covered Totals

Molts

No molts

18 14 32

37 12 49

Molts

No molts

13 10 23

females were resubmersed

to air.

Totals 55 26 81 x: = 1.558, P > 0.05 Totals

I 2 9 Fisher’s

* l-day exposed

by water vs. tubes exposed

exact probability

on Day 7 and, therefore,

20 12 32 test P = 0.242

were omitted

from this comparison.

REPRODUCTIVE

Timing

ofmolts

BEHAVIORS IN MICRODEUTUPUS

relative to water retention

GRYLLOTALPA

123

in tubes

Even though water was poured out of the dishes, most of the 39 tubes retained a droplet of water (Table IV; 28 of 39, x: = 7.410, P < 0.01). Water remains inside the tube because the ends of the tube collapse when water is decanted from the dish. Tubes TABLE IV Fates of 39 females whose tubes were examined for water-droplet retention during exposure periods. Tubes

Die on exposure day Molt on exposure day Molt after exposure day Totals

With droplets

Without droplets

4 14 IO 28

6 0 5 11

that retained water were sausage-shaped and the females’ respiratory pleopods moved freely inside. In contrast, tubes without water were flattened, and their walls adhered to the females’ body. Female’s pleopod movements were much reduced. 10 of the 39 females died but the absence of water did not increase mortalities (four of these female’s tubes retained a water droplet and six did not). The remaining 29 females molted, either during days of exposure (141, or after resubmersion (15). Each of the tubes of the females who molted during periods of exposure contained water. But none of the five females whose tubes were dry during exposure days molted during exposure; they all molted after they were resubmersed. The difference was significant (0 of 5 vs. 14 of 24 females molted during exposure periods, dry tubes vs. wet tubes, respectively; Fisher’s exact probability test, P = 0.03). Thus, (1) most of the tubes (83 %) retained a water droplet even though all water was decanted from their dishes; and (2) the only females that molted during exposure periods were those whose tubes retained water.

DISCUSSION I. ROLE OF TUBE AS A STIMULUS FOR MALE-REPRODUCTIVE

BEHAVIORS

The results of these observations suggest that cooccupancy of a tube constructed by an amphipod is a prerequisite for the expression of males IPBs. The importance of cooccupancy was established by the observation that although in contact with the tube, if animals were outside it IPBs were not expressed. The importance of an ~phipod tube was established by the observations that IPBs are generally not expressed when glass capillary tubes or unmodi~ed Ulva fucfuca thalli alone were present, but were expressed if the capillary tube containing a female was placed inside a male M. grytlotal’a tube.

B. BOROWSKY

124

In contrast to what was expected, however, the type of ~phipod that eonstru~ted the tube seemed to have fittle effect on the expression of IPBs. Thus, receptive females do not mark their tubes. II. ROLE OF TUBE

IN PROTECTING

AGAINST

EXPOSURE

The data suggest that amphipods that reside in tubes are protected from desiccation. Lengthy exposure periods increased mortality and delayed molts but the overall effects of exposure to air were surprisingly benign. Most females survived and molted even when exposed for 2 days. The molt cycle is driven principally by hormones but the length of the intermolt period can be modified by a variety of physiological and environmental factors (Skinner, 1985). The present study shows that exposure is another env~onment~ factor that can delay the molt. When water was decanted from the dishes, some tubes retained a droplet of water [also noted for the tanaid Tanais cavoli~ii by Johnson & Attramadal (1982)]. In the present study, females whose tubes retained the droplet molted at about the same time as females residing in dishes from which water was not removed. But females whose tubes did not retain the droplet molted only after resubmersion [as in Gammarus palustris (Borowsky, 1980b)]. Ecdysis may be restricted to periods of submersion because desiccation occurs faster through new rather than older calcified exoskeleta. In addition, water may be required for molting either for buoyancy or lubrication. III.

ECOLOGICAL

IMPLICATIONS

OF RESULTS

Exposure to the air may have little impact on the animals in the field - as long as they are inside their tubes. First, exposure periods are shorter in the field than they were in this experiment; second, the sediment probably remains wetter; and third, the shapes of the tubes may enhance water-droplet retention. The shapes of the tubes vary with the substratum: if the animal builds its tube on a hard surface, the tube is most often horizontal (as they were in the present study). But if the animal builds in sediments B 1 cm deep, it is shaped into a vertical U, with the two entrances opening onto the surface of the sediment (Dewitt, 1985; pers. obs.). The U shape forms a cup which captures a droplet of water as the tide recedes, The results support the hypothesis that the tube stimulates reproductive behaviors because the tube enhances survival. Reproductive behaviors were only expressed by males while inside amphipod tubes. The importance of residence inside a tube is that, although the tube itself may be exposed, if the tube retains a droplet of water, the animals, in effect, are not exposed. The importance of the amphipod tube is that the walls of the tube are elastic. As water recedes, the ends collapse, thus enhancing the retention of water. The U shape helps retain water too. Thus, it is adaptive for a male

REPRODUCTIVE

BEHAVIORS

IN MICRODEUTOPUS

GRYLLOTALPA

125

to express reproductive behaviors to females inside amphipod tubes to ensure the survival of his offspring. It is possible that tubes serve additional functions in M. grylZota@a as well. For example, it is likely that they afford protection against important visual predators such as fish (Chapman et al., 1981). The greater the degree of protection the tube affords, the greater the selective pressure to behave in a way that results in one’s offspring remaining inside them.

REFERENCES Borowsky, B., 1980a. The physiological control of reproduction in Microdeutopus gryllotalpa (Crustacea: Amphipoda). I. The effects of exogenous ecdysterone on the female’s molt and behavioral cycles. J. Exp. Zool., Vol. 213, pp. 399-403. Borowsky, B., 1980b. Factors that affect juvenile emergence in Gammaruspalustris (Bousfield, 1969). J. Exp. Mar. Biol. Ecol., Vol. 42, pp. 213-223. Borowsky, B., 1983a. Behaviors associated with tube-sharing in Microdeutopus gtyllotulpa (Costa) (Crustacea: Amphipoda). J. Eq. Mar. Biol. Ecol., Vol. 68, pp. 39-5 1. Borowsky, B., 1983b. Reproductive behavior of three tube-building peracarid crustaceans: the amphipods Jassa fulcura and Ampithoe vulidu and the tanaid Tanais cavolinii. Mar. Biol., Vol. 11, pp. 251-263. Borowsky, B. & R. Borowsky, 1987. The reproductive behaviors of the amphipod crustacean Gummurus pulustris (Boustield) and some insights into the nature of their stimuli. J. Exp. Mar. Biol. Ecol., Vol. 107, pp. 131-144. Chapman, J.C. Onuf & M. Quammen, 1981. Effects of fish predation on peracarid crustaceans in Mugu Lagoon, California. Washington Sea Grant publication, University of Washington, Seattle, Washington. Christofferson, J. P., 1978. Evidence for the controlled release of a crustacean sex pheromone. J. Chem. Ecol., Vol. 4, pp. 633-640. Crane, J., 1975, Fiddler crabs of the world (Ocypodidae: genus Uca). Princeton University Press, Princeton, New Jersey, 736 pp. Dewitt, T.H., 1985. The behavior and ecology of migration and colonization in the epibenthic, tubicolous amphipod Microdeufopus gryllotalpa. Ph.D. thesis, State University of New York at Stony Brook. Eales, A. J., 1974. Sex pheromone in the shore crab Curcinus maenus, and the site of its release from females. Mar. Behuv. Physiol., Vol. 2, pp. 345-355. Gleeson, R. A.,1980. Pheromone communication in the reproductive behavior of the blue crab Callinectes sapidus. Mar. Behav. Physiol., Vol. 7, pp. 119-134. Johnson, S.B. & Y.G. Attramadal, 1982. A functional morphological model of Tanais cavohnii Milne-Edwards (Crustacea, Tanaidacea) adapted to a tubicolous life-strategy. Sursia, Vol. 67, pp. 29-42. Salmon, M., 1983. Courtship, mating systems, and sexual selection in decapods. In, Studies in adaptation, The behavior in higher Crnstuceu, edited by S. Rebach & D. W. Dunham, John Wiley & Sons, New York, pp. 143-170. Skinner, D. M., 1985. Molting and regeneration. In, Integument,pigments and hormonalprocesses. The biology of the Crustacea, Vol. 9, edited by D. Bliss & L. Mantel, pp. 44-146.