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1 Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425
2 Department of Biological Sciences, George Washington University, Washington, DC 20052
* To whom correspondence should be addressed. E-mail: grossp{at}musc.edu
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Morphogenesis and molting history can be affected by environmental factors such as diet, temperature, and photoperiod (reviewed in Knowlton, 1974), and by hormonal cues. An "X-organ–sinus gland complex," located in each of the paired eyestalks just medial to each optic complex, has long been known as a source of many hormones that can affect various physiological processes either directly or indirectly in adult decapods (reviewed in Carlisle and Knowles, 1959; Passano, 1960, 1961; Welsh, 1960; Kelly, 1967; Jenkin, 1970; Kleinholz, 1976; Cooke and Sullivan, 1982; and Fingerman, 1987). This complex also plays a role in control of larval development (reviewed in Christiansen, 1988; Charmantier and Charmantier, 1998). Eyestalk ablation has been shown to produce supernumerary larval stages, larval-postlarval intermediates, or both in various crustaceans such as Rhithropanopeus harrisii (Costlow, 1966) and Homarus americanus (Charmantier et al., 1985; Charmantier and Aiken, 1987).
Larvae of the snapping shrimp Alpheus heterochaelis Say from coastal North Carolina (described by Knowlton, 1973) exhibit some unique developmental features. The larval phase is abbreviated, lasting only 4–5 days when reared at 22–25°C and consisting of three larval (zoeal) and one postlarval stage. The larvae hatch with a large amount of stored yolk and oil, which they apparently use as the sole source of energy throughout the larval period. Exogenous feeding does not begin until the postlarval stage when the mouthparts develop into functional feeding organs (Gross and Knowlton, 1991).
Eyestalkless A. heterochaelis larvae have been observed to form larval-postlarval intermediates consistently as long as eyestalk removal is performed prior to the midpoint of larval Stage III (Knowlton, 1988, 1994). An array of different morphologies was discerned, depending on the time (during the second and third larval instars) that the eyestalk extirpation was performed. Knowlton (1994) described four types of larval intermediates, designated stages IVA through IVD, each possessing more developmentally advanced features and occurring between stage III and the postlarva. In his study, operations were performed once a day at the same time of day over the 4- to 5-day larval period. In the present study, this protocol was refined by increasing the number of time points at which operations were performed to seven (i.e., at 2 h, at 20 h, and at subsequent 10-h intervals over the duration of larval development), utilizing more animals at each time point, and maintaining the animals over several instars (molts). Two series of experiments, each involving larvae hatched from eggs borne by four different females, were executed. Specific objectives of these experiments were (1) to ascertain whether morphogenesis occurs in discrete steps or is continuous and (2) to establish if extirpation at various times results in developmental arrest (either complete or delayed), and whether there is a critical threshold time after which development to the postlarval stage is determined.
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Rearing of larvae
Seawater used in all experimental procedures was prepared as described in Gross and Knowlton (1997). Hatched zoea larvae were maintained in UV-treated seawater, which was further filter sterilized through a 0.45-µm filter to which the following chemicals were added: 50 ng/ml amphotericin B, an antifungal agent, plus 50 µg/ml each of the antibiotics streptomycin sulfate and ampicillin (ICN Biochemicals, Costa Mesa, CA). Larvae were maintained in 10 ml of processed seawater, changed daily, in an incubator at a constant 25°C to provide optimum development and a "standard" 4-day larval development pattern for control animals (Knowlton, 1973). When animals reached the postlarval and juvenile condition, they were fed freshly hatched Artemia salina (brine shrimp) nauplii ad libitum. General laboratory procedures are given in Gross and Knowlton (1997, 1999).
Protocol of operations
Immediately after all larvae in a particular brood had molted to larval stage II (about 2–3 h post-hatching), a subset of the brood, designated t2 (for 2 h post-hatching) was subjected to eyestalk extirpation. A second population was ablated 18 h after t2 and designated t20. Eyestalk ablations were then performed at 10-h intervals after t20 up to 70 h, with groups being designated accordingly. In a previous study, it was found that larvae ablated at 10 h post-hatching (t10) closely resembled those of the t20 group (data not shown; Gross and Knowlton, 1991). Typically, eyestalk ablations at t2 and t20 occurred during Stage II and all other ablations during larval stage III, with t30 occurring almost immediately after the molt to stage III. A number of larvae in each brood were retained as unoperated controls. The control group and each group of larvae designated for surgery at a particular time point had roughly equal numbers of animals.
Analysis of intermediate morphologies
In the first series of experiments, a total of 274 larvae from four broods were used. After the final set of operations at t70, each larva was individually monitored as it molted to the intermediate form (stage IV of Knowlton, 1994) or postlarva (in controls). Data pertaining to size differences are given in Gross and Knowlton (1999). At this time, the animal was preserved in 70% ethanol (EtOH). Each larva was then cleared in 5% KOH, stained using carmine-borax/35% EtOH, and placed in 100% glycerol by serial transfer, in preparation for structural examination and measurement (Guyer, 1906; Gross, 1995). Glycerin was chosen as the final medium because it maintains the clarity of the stained specimen; it is significantly more viscous than water, giving the specimen a greater internal rigidity; and it causes the otherwise brittle exoskeleton to become pliable, allowing the appendages to be moved and posed easily during analysis of larval characteristics. Both dissecting and compound microscopes were used to analyze morphological features of the fourth and fifth instar intermediate forms with respect to time of ablation, and were related to analogous morphological characters found in control postlarvae and juveniles.
In the second series of experiments, a separate set of 629 larvae was used. The procedure was similar to that already outlined except that the larvae were not sacrificed but were allowed to continue development through subsequent instars (molts). For consistency, operations on larvae were performed at the same time points. As they developed, live animals were examined for morphological diagnostic characteristics (along with instar duration and mortality; see Gross and Knowlton, 1997). Each larva was assigned a substage or "form" designation based on the system proposed by Knowlton (1994). Two behavioral characters were also recorded after each molting: (1) the orientation of the larva—dorsal side up (walking posture), consistent with an adult benthic life habit, or dorsal side down (swimming behavior), exhibited by pelagic larva; and (2) consumption of Artemia salina nauplii, as evidenced by food (opacity) in the stomach.
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In Knowlton’s (1994) stage IVC, the head and abdominal regions are morphologically postlarval, but the thoracic appendages are only partially developed toward the postlarval form. Like the previous intermediate forms, the IVC form generally swims ventral side up and is incapable of feeding. In the present study, three additional IVC types, based on the degree of morphological advance in the thorax and abdomen, were distinguished (Table 1).
Stage IVB/C larvae, found only among animals ablated at t30 (Table 2), exhibited most of the characteristics of Knowlton’s stage IVC, but only the exopod of pereiopod 4 showed reduction in size and loss of swimming function. Animals designated stage IVC1 lacked the pair of spines on the dorsal side of the telson about three-quarters of the length distal from the joint connecting the telson to the sixth abdominal somite (as in the postlarva), whereas those designated stage IVC2 possessed these spines. In addition, the IVC1 form was more commonly found in animals from the t30 group (relative to the t40 group). The IVC2 form was more common than the IVC1 form among larvae ablated at t40 (Table 2).
Some characteristics were shared by all larvae classified as stage IVC. For example, although the uropodal exopodite is jointed in the postlarva, it was never jointed in animals classified as stage IVC; however, the typical postlarval spine located on the outer margin was always acquired. The antennal flagellum was essentially postlarval in its length and number of segments. In some animals, the inner and outer antennular flagella had 3 segments instead of the postlarval 4. The antennular peduncle was always 3-segmented and the basicerite was usually present, but the statocyst was never completely formed.
The thoracic appendages of the stage IVC form (both IVC1 and IVC2) were in many ways still similar to those of earlier intermediate forms, and morphogenetic advance was more pronounced in posterior thoracic appendages than in the anterior ones. The exopods of pereiopods 1 and 2 exhibited almost no change relative to stage III. However, those of pereiopods 3 and 4 were dramatically shortened (but not to the extent seen in the normal postlarva) and curled in appearance, with 4–6 misshapen setae on each terminus. Pereiopod 5, which lacks a swimming exopod throughout development, remained 7-segmented, but its dactyl was blunter than the very long styliform appendage associated with Stage III. All joints on this appendage appeared to be functional, with the joint between the merus and carpus modified to form a knee-like joint more suited to walking. The endopods of pereiopods 3 and 4 underwent proportionately more morphological change (compared with those of pereiopods 1 and 2). In most animals, they had the full complement of 7 segments (sometimes 6), with the styliform dactyl being replaced by a blunter form; joints on these appendages were always functional. The endopod of pereiopod 2 displayed the most segments (8–10), but development was incomplete as the joints were not articulating. New segments were usually added to the endopod of pereiopod 1, but the joints of this appendage were never fully articulated at this stage.
Individuals assessed as stage IVD (Knowlton, 1994), as exemplified by larvae ablated at t30–t60 (Table 2), possessed most of the characteristics of the postlarva while still retaining a few larval characteristics. In these animals, the telson was postlarval in shape, possessing the typical 4 + 4 terminal setation pattern and the characteristic pair of spines located on the dorsal surface. The uropods were fully developed, with the exopodite bearing both the outer spine and distal joint. The antennal flagellum was as long as that of the typical postlarva, with a full complement of segments (22–26). Some specimens possessed 3-segmented inner and outer antennular flagella (rather than the usual 4 segments), but the peduncle was always 3-segmented, with basicerite and statocyst fully developed. These larvae exhibited complete transformation of pereiopods 3–5 to postlarval form. The endopod of pereiopod 1 was morphologically postlarval in this stage as well, always being composed of the full complement of 6 segments, with the elbow-like joint between merus and carpus, and completion of the joint between the propodus and dactyl to form a functional chela. The endopod of pereiopod 2 still showed some variability in segment number (9–10), but was functionally complete, with an articulated chela and an elbow-like joint between the merus and first carpal article. Exopods of pereiopods 3 and 4 were usually devoid of setation and nub-like (Table 1), as in the postlarva; in some specimens, however, these exopods were longer than that of a normal postlarva. Exopods of pereiopods 1 and 2 were not functional but had not attained the naked nub-like appearance of the postlarva. Stage IVD animals typically walked ventral side down, like the postlarva, consistent with the presence of a fully developed organ of balance (statocyst). They were usually capable of feeding (also like the postlarva), as evidenced by the presence of Artemia salina nauplii remains in the stomach.
A few animals (C/D in Table 2) ablated at t30 and t40 possessed setose exopods on pereiopods 1–3. Similarly, a significant proportion of those larvae ablated at t50–t70 possessed setation only on the exopod of pereiopod 1 (D/PL). These larvae appeared to be similar to the postlarva in other respects, except that the second pereiopodal endopod occasionally had 9 segments rather than 10.
In summary, detailed examination of ablated larvae from each time point yielded in essence a continuum of stage IV morphologies between larval stage III and the normal postlarva, and larvae ablated at t30 and t40 showed the greatest variability in intermediate form morphology (Table 2).
Analysis of fifth instar morphology
Intermediate morphologies encountered at the fifth instar (i.e., after four molts post-hatching), which is normally the molt to a juvenile form, are enumerated in Table 3 with the same nomenclature used to describe fourth instar forms. A stage V designation identifies fifth instar intermediates, which are still morphologically intermediate between larval stage III and postlarva, while E, F, and G designations are reserved for individuals with characteristics intermediate between the postlarva (PL) and juvenile (J).
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Many (45.0%) of the larvae ablated at t30, which as fourth instars formed a wide array of larval-postlarval intermediate forms (Table 2), molted to the juvenile form at the next molt; nine animals (15.0%) resembled normal postlarvae at the fifth instar (Table 3). A significant number of animals from this group exhibited characteristics, noted under E in Table 1, that were intermediate between the normal postlarva and juvenile. Some larvae ablated at t30 were found to possess the juvenile telson morphology with vestigial exopod nubs on pereiopods 1–3, or on 1–2 only (remaining pereiopods being uniramous); these intermediate forms were designated F and G, respectively (Table 3). Of the broad spectrum of intermediate stage IV types noted among larvae ablated at t40 (Table 2), the majority (70.5%) molted to juvenile at the fifth instar, but most of the others (E in Table 3) retained all pereiopod exopod rudiments on pereiopods 1–4. All larvae from the t50–t70 groups molted to the juvenile form at the fifth instar.
All animals surviving to the sixth instar, which attained some semblance of postlarval morphology at the fifth instar, were found to exhibit morphology consistent with classification as juveniles. Comparing the distribution of larval intermediates found at the fifth (vs. fourth) instar, larvae ablated prior to the molt to stage III (t2 and t20) appeared, for the most part, to be incapable of completing development (metamorphosis). Conversely, those ablated after the molt to stage III (t30 through t70) completed metamorphosis by the sixth instar. However, animals ablated at t30 (and to some extent at t40) showed a definite lag in morphogenesis relative to larvae ablated at later times and were more prone to appear as postlarval-juvenile intermediates during the fifth instar.
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Crucial points in morphogenesis
The crucial point at which the A. heterochaelis larva becomes capable of completing morphogenesis to the juvenile condition (normally the fifth instar) seems to occur around the time of ecdysis to stage III. None of the larvae in the t2 group changed structurally beyond form B (Table 2), and most of the t20 animals died without morphological advance beyond form B/C (Table 3). A small number of animals from the t20 group did succeed in attaining a postlarval morphology, or a form close to it (VD/PL/J), at the fifth instar, but only two individuals survived to molt to a morphogenetically normal juvenile at the sixth instar. Thus, with few exceptions, larvae ablated before the molt to stage III were incapable of morphogenetic advance to the postlarval or juvenile form. Similarly, in Homarus americanus (which exhibits a pattern of larval development similar to A. heterochaelis), Charmantier and Aiken (1987) noted that the critical period for eyestalk removal leading to developmental arrest and the appearance of larval-postlarval (stage IVa) intermediates was in molting stage D1 of larval stage II. Conversely, A. heterochaelis larvae that underwent eyestalk ablation during stage III could complete morphogenesis to a feeding juvenile or could attain an intermediate morphology between postlarva and juvenile (i.e., forms E, F, and G) during the fifth instar. However, a few animals ablated close to the completion of ecdysis to stage III did not advance beyond form B. Thus, the "turning point" separating completion of metamorphosis from developmental arrest seems to be at the interface between stages II and III at or about the time of ecdysis, which is in agreement with the findings of Knowlton (1994).
Whereas Knowlton (1994) did not note any further morphological changes among larvae ablated during stage II (before attaining the IVA or IVB form), our more detailed analysis showed that by the fifth instar most larvae exhibited some change in morphology relative to their stage IV counterparts. The majority of t2 animals advanced from form A1 to the A2, A/B, or B form, and most t20 animals surviving to a fifth instar also advanced slightly in form. Thus it appears that even though metamorphosis is blocked by ablation before the third instar, morphogenesis is not completely arrested. Even among larvae that can complete metamorphosis to the juvenile form, the effects of eyestalk ablation may only be overcome over multiple instars and elongation of the larval period (Gross and Knowlton, 1999). On the other hand, even though t30 and t40 larvae were able to complete metamorphosis to a morphologically normal juvenile by the sixth instar, there was a definite lag in morphogenesis.
Form D animals are substantially different and more advanced than those of form C since they possess fully functional (articulating) thoracic endopods (as in the postlarva) and are able to feed, orient themselves ventral side down, and walk. It is apparent that at about 35–40 h post-hatching the third instar larva attains the ability to make, after molting to the fourth instar, a more "complete" morphological conversion from a planktonic larval existence to a more adult benthic existence. Ablation after about 35 h post-hatching more frequently resulted in the stage IVD form or more advanced forms (up to and including normal postlarva—Table 2). Most t50 individuals exhibited morphology consistent with the postlarva (at the fourth instar). But morphogenesis was not "finalized" until late in the third instar, since larvae with form D features were seen even among t70 animals.
The nature of the morphogenetic continuum
Carlisle and Knowles (1959) postulated that factors located in the X-organ and released at the sinus gland control molting and that morphogenesis is a stepwise process which coincides with the molt cycle and may even be controlled by it. This does not appear to be the case for A. heterochaelis larvae, which, when their eyestalks are ablated at various times, display a continuum of intermediate morphologies independently of the molting cycle. If molting and morphogenesis were inextricably tied, then it would be reasonable to assume that premature molting would either fail to produce any morphogenetic advance or would yield a stepwise array of developmental intermediates. Knowlton (1994) observed a limited array of intermediates in experiments in which eyestalks were ablated only once per day. However, by increasing the number of time points for eyestalk ablation, a more continuous array of intermediates was produced that encompassed many more intermediate morphological forms. Our results argue against developmental patterns in higher Crustacea being "fixed" (Gurney, 1939), with all individuals showing exactly the same changes at each molt; or "compartmentalized" (Fraser, 1936), with individuals exhibiting morphological variation within a limited range (compartment). Indeed, they provide ample evidence that development in these crustaceans is continuous—a gradual process in which different body segments attain the potential for differentiation into their adult morphology at different times. Removal of the eyestalk at intervals makes this aspect of gradual morphogenetic change more apparent and shows that molting and morphogenesis are concurrent but independently controlled processes.
The axis of morphogenetic change
Eyestalk ablation at various times interrupts the normal process of morphogenesis at different points along the morphogenetic continuum (described above). Differentiation appears to take place at different rates in each segment in a "double gradient" pattern. This is well evidenced by the descriptive data collected from groups of animals at each ablation time point. The ability to differentiate at the next molt is attained in the tail region first, with uropodal endopods and pleopods gaining the ability to differentiate to postlarval form (and size) as early as 2 and 20 h post-hatching, respectively. Under eyestalk control, ability to differentiate is attained in the head region slightly later, since larvae ablated at t20 and t30 begin to exhibit morphogenetic advances (changes in form and size of appendages) in the head region at the fourth instar. The segment bearing pereiopod 5 gains the ability to metamorphose (i.e., convert the styliform dactyl to a blunter form and shorten in overall length) at the fourth instar at about the same time as the head appendages (around t30), reinforcing the idea that determination is advancing forward from the tail. Among eyestalkless animals, determination in the middle region (thorax) occurs later than in the head and tail region and is attained in exopods of the third and fourth pereiopods well before those of the first and second.
Differentiation in form (e.g., adding segments to appendages) precedes the potential for functional changes (e.g., formation of movable joints). Pereiopod 5 differentiates to postlarval length and segment number with the t30 group, but functional joints are not commonly found on this appendage at the fourth instar in these animals, whereas in the t40 group the fifth pereiopod is usually fully functional by the fourth instar. Among larvae ablated at t30, the third and fourth pereiopodal endopods were equivalent to controls in length and segment number, but functional joints on these appendages were generally not seen until later (e.g., among t40 or t50 animals). At the fourth instar, endopods of pereiopods 1 and 2 both become functional among larvae ablated around t40 (or t50). They lag behind pereiopods 3–5 in onset of morphogenesis (reaching postlarval length and segment number) and attainment of functionality (articulating joints).
One can hypothesize that, under normal circumstances, the ability to metamorphose (at the molt to the fourth instar) is attained at different times by different body regions and even by specific segments within a body region of the A. heterochaelis larva. Factors in the eyestalk either control this process directly, or different body segments become receptive at different times to a factor or factors released by the eyestalk. Morphogenetic advance is not manifested until ecdysis, but the potential for morphogenesis toward the postlarval form starts early in the second instar, increasing gradually and continuously over the larval period. This potential for morphogenesis (differentiation at the next molt) is not the same along the posterior-anterior axis but instead may be visualized as a "double gradient" extending from the posterior and anterior ends toward the thorax. The trend is that the capacity to differentiate develops in the abdomen and abdominal appendages (uropods and pleopods) first, followed shortly thereafter by the head and its appendages, and lastly by the thorax. In the thorax, determination occurs earliest in the segments bearing pereiopod 5 and maxilliped 1, while the last segment to be determined bears the second pereiopod. Thus, the double gradient simultaneously extends forward from the fifth pereiopod and backward from the first maxilliped, converging on the second pereiopod.
Possible mechanisms of hormonal control
Several models could account for the production, through bilateral eyestalk ablation, of larval-postlarval intermediate forms and for the double gradient pattern of gradual determination. One explanation is that eyestalk removal deprives the developing larva of a critical regulatory principle or hormone that causes a given segment to transform from larval to postlarval morphology, and that this potential is realized gradually or at different times in different segments. The functional details of these mechanisms are still a matter of conjecture.
The simplest explanation for the appearance of larval-postlarval intermediate forms, advanced by Knowlton (1994), posits the existence of a morphogenetic hormone (MH) that is secreted by the eyestalk (presumably by the X-organ–sinus gland complex). This release would necessarily be gradual and begin early in stage II. Imam (1982) noted that certain tissues having a glandular appearance early in stage III differentiate in the larval eyestalk, only to degenerate in the postlarval eyestalk, surmising that these structures play a secretory role in the control of morphogenesis. A morphogenetic hormone would have to activate different segments at different times: either a segment could respond when the hormone reached a specific (and variable from segment to segment) concentration threshold in that segment, or it could respond only at a specific time in the morphogenetic continuum. If morphogenetic activation of certain segments does depend on a certain MH threshold, then removal of the eyestalk (which deprives the developing larva of any further production of this hormone) should produce morphogenetic arrest in those segments—presumably the more central thoracic ones—that were below threshold at the time of eyestalk removal. This condition should be permanent for those segments below threshold, since circulating titers of the morphogenetic factor would be below threshold level at the time of ablation. Clearly this is not the case since almost all larval-postlarval intermediates produced by means of bilateral eyestalk ablation during stage III are capable of metamorphosis to a normal form, given enough time.
If specific segments became responsive to MH at different times during the morphogenetic continuum, then in order for morphogenesis to continue in those segments that are activated later or at a slower rate, MH would have to be available in the circulation well after its source was removed. MH would thus have to be an extraordinarily long-lived factor, which is not typical for circulating hormones. Alternatively, MH may be released in small amounts during stage II (enough to instigate abdominal morphogenesis even after eyestalk ablation), but this release is either not adequate to activate other body regions or other body regions are unresponsive at this stage. After the molt to stage III, MH may be released in a quantity high enough to instigate morphogenesis in all body regions but in a quantity too low to control the timing of morphogenesis (since larvae ablated at t30 exhibit a wide variety of larval-postlarval intermediates but can eventually attain the normal juvenile form), especially in the thoracic region. That is, circulating titers of MH may control the timing of morphogenesis in each segment, but activation depends on a certain basal concentration of MH being released.
In insects, morphogenesis is not controlled by a hormone that instigates the process but by a metamorphosis-inhibiting factor (MIF) or juvenile hormone (JH) that inhibits its onset until a specific time (pupation). This hormone (JH) is maintained in high titers during the larval phase and drops precipitously at metamorphosis. If the control of metamorphosis were similar in the Crustacea, it seems more likely that the eyestalk produces some sort of regulatory factor, which controls morphogenesis through a second hormone.
Some inferential evidence for a crustacean juvenile hormone or metamorphosis-inhibiting factor does exist. Freeman and Costlow (1983) found that whole-body extracts of stage III Rhithropanopeus harrisii larvae inhibited resorption of the large dorsal spine (characteristic of the zoea) in vitro. Further, Laufer and Borst (1988) have advanced the idea that methyl farnesoate (MF), produced in the mandibular organ (MO), functions as a MIF; they established that control of its secretion is under the influence of tissues in the eyestalk. It has been shown conclusively that the X-organ–sinus gland complex secretes a mandibular organ inhibiting hormone (MOIH), which controls titers of MF by inhibiting its production within the MO (Liu and Laufer, 1996; Wainwright et al., 1996; Liu et al., 1997)
The results of the present research cannot conclusively distinguish between Knowlton’s "MH hypothesis" and the existence of a MIF, and it may be that both systems exist in tandem. Knowlton (1994) asserted that in such a case it would be a balance between MIF and MH that controls the onset and timing of morphogenesis. But on the basis of this and other research (e.g., Freeman and Costlow, 1983), it is possible that one or more factors, released from the eyestalk, inhibit MIF function or release from a second gland, and that different body segments have unique thresholds to MIF titers. The hypothesis that the eyestalk produces a regulatory hormone modulating the release of MIF would partially explain the double gradient pattern of morphogenetic potentiation in A. heterochaelis larvae if, as postulated by Borst et al. (1987), the mandibular organ is the source of MIF and that MIF is in fact MF. Simple proximity to this gland could be related to circulating titers of MIF. The extreme ends of the animal could be subjected to lower amounts of MIF naturally, by virtue of being the most distant from the mandibular organ, and thus would be released from inhibition earlier, while the central body segments would be proportionately more affected by MIF and released from inhibition later as titers of MIF dropped off under the influence of eyestalk secretion. Release of MIF and circulating titers may be controlled by a factor released from the X-organ–sinus gland complex in the intact animal. In eyestalkless animals, responsiveness to MIF may drop over time (even though circulating titers may not), or circulating titers of MIF may fall even without the influence of the factor or factors released by the eyestalk (though more gradually than in intact animals). This could produce the double gradient effect as each segment becomes progressively less responsive or titers fall below the unique threshold of each segment sequentially.
The idea that MF is the active JH in crustaceans is still very much in doubt, but some compelling evidence does exist. Exposure to MF has been shown to retard metamorphosis in H. americanus (Borst et al., 1987), to retard early development in Macrobrachium rosenbergii (Abdu et al., 1998a), and to have a juvenilizing effect on Balanus amphitrite larvae (Smith et al., 2000). However, as pointed out by Abdu et al. (1998b), these effects could be due to nonspecific toxicity related to continuous exposure to MF. These same authors (Abdu et al., 1998b) present a strong case that MF is the direct hormonal agent controlling metamorphosis; the arguments are particularly convincing when coupled with the identification of an eyestalk-related MOIH. In relation to these studies, the present work clearly indicates that MIF, be it MF or some other factor, controls metamorphosis directly and that the relationship between morphogenesis and molting is not one that affects molt timing but one that affects hormonal activation of the remodeling process itself.
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