Reproduction
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  Settlement & metamorphosis
  Topics on reproduction include settlement & metamorphosis considered here, and SPAWNING, BROODING, LARVAL LIFE, and EARLY JUVENILE considered elsewhere.
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Research study 1
 

drawing of anterior end of a tunicate tadpole larvaThe tadpole larvae of tunicates have adhesive papillae at the front that secrete a sticky attachment substance.  Once attached, metamorphosis is quick.  Within minutes the tail begins to be resorbed, the larva rotates to bring the siphonal openings into correct adult orientation, and the branchial-basket filtering system becomes functional.  In addition to the tail being resorbed, other structures useful in swimming and navigation in the larva are resorbed or reduced in size.  In general, there are 2 main groups of structures in the larva, one related to the larva; the other, to the adult:

1) transitory larval organs: notochord, dorsal tubular nerve cord, tail, adhesive papillae, and various ganglia and sensory organs (e.g., ocellus or eyespot).  These structures (coloured purple in the drawing) function in swimming, sensory input, and attachment, and are lost or resorbed during metamorphosis.

2) prospective juvenile/adult organs: siphons, branchial basket, endostyle, ampullae, gut, cerebral ganglion, and heart .  These structures (coloured yellow) are in an arrested state of development in the larva, and become functional shortly after metamorphosis.  Drawing of the colonial tunicate Distaplia occidentalis larva from Cloney 1982 Am Zool 22: 817.

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Research study 2
 

photograph of colonial tunicate Distaplia occidentalisDistaplia occidentalis is a colonial tunicate with arborescent growth-form.  Its tadpole larvae are large, even among the relative giants seen in tunicates, and reach over 3mm in length.  In laboratory studies at Friday Harbor Laboratories, Washington the larvae attach to the substratum in about 30sec (at 15°C) and metamorphose quickly.  Within 7min the tail is mostly resorbed and the size of the metamorphosing tunicate is reduced to about one-fifth of what it was at the start.  Walters & Wethey 1991 Biol Bull 180: 112.

NOTE  lit. “tree becoming” L., referring to the spreading growth-form of the colony

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Research study 3
 

series of drawing showing metamorphosis in a tunicateMetamorphosis transforms the non-feeding, motile tadpole larva into a filter-feeding attached juvenile, and thus involves numerous morphological and physiological changes.  These changes are initiated at settlement.  Some, such as papillary eversion (for attachment) and tail resorption, take seconds or minutes; others, such as completion of rotation, may take hours.  Major events that occur from first contact with the substratum are eversion of the papillae and secretion of adhesives, while later events include retraction of these papillae, tail resorption, rotation of organs through 90o, expansion of branchial basket and tunic, and resorption of ganglia and eyespot (ocellus). For visual reference in the drawings of metamorphosis the branchial basket is coloured yellowish.  Drawings of metamorphosis of Distaplia occidentalis larva from Cloney 1978 p.255 In, Settlement and metamorphosis of marine invertebrate larvae (Chia & Rice, eds) Elsevier, NY; Cloney 1982 Am Zool 22: 817.

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Research study 4
 

illustration of a solitary tuniate Chelyosoma productumphotograph of a larva of the tunicate Chelyosoma productumMany tunicate species settle gregariously with members of their own species.  In some cases this owes to the extremely short swimming period of the larva, so that settlement is close to the adults that produce the larvae, but other species such as the solitary tunicate Chelyosoma productum in the San Juan Islands, Washington appear to actively seek out and settle near other conspecific juveniles, or on the tunics of conspecific adults.  In the field, Chelyosoma forms clumps of all sizes under floating docks.  Tests of settling behaviour in the laboratory show that the larvae of C. productum are mostly indifferent to light.  In one set of tests, where 10,000 competent larvae are allowed to settle in Petri dishes that are half shaded and half lighted, 53% settle on the shaded sides, while 47% settle on the lighted sides.  Intrestingly, under crowded conditions the newly settled larvae produce what the authors term “ampullae”.  These are fluid-filled evaginations from the epithelium, usually 3-4 in number, that distend the tunic and create more space for the larva (see photograph on Right). After about 10d (at 11°C) following tail resorption, these “primary” ampullae are resorbed and a larger, secondary ampulla is produced posteriorly.  This creates an elongated form to the zooid and tends to push the siphons out into the water column, perhaps for better access to food.  By 18d a crowded zooid’s ampulla comprises about two-thirds of its body, while an uncrowded zooid lacks an ampulla entirely.  Gregariousness does not function for reproduction because C. productum is a self-fertilising hermaphrodite.  The authors note that this is the first description of gregarious settlement in an ascidian. Young & Braithwaite 1980 J Exp Mar Biol Ecol 42: 157; drawing of Chelyosoma productum modified from Morris et al. 1980 Intertidal invertebrates of California. Stanford Univ Press, Stanford.

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Research study 5
 

Most ascidian larvae exhibit a characteristic shadow response, in which they swim in response to a sudden decrease in light intensity.  The behaviour is usually assumed to help the larvae locate shaded sites on which to settle.  Experiments at Friday Harbor Laboratories, Washington to test this idea use 8 species of tunicates, and involve offering tadpole larvae the choice of shaded and unshaded surfaces in up and down orientations after being cultured in photograph of several tunicates Cnemidocarpa finmarkiensis in an old clam shellconditions of continuous light, continuous dark, or alternating light/dark.  The tadpoles are induced to swim by a shadow and then their settlement locations tallied. Results show only one or two significant preferences overall by the tadpoles for certain substrata, and in no consistent pattern.  The authors conclude that the mechanism by which tadpoles locate shady areas to settle and metamorphose is independent of the shadow response.
Young & Chia 1985 J Exp Mar Biol Ecol 85: 165.

NOTE  the 8 species are Corella inflata, C. willmeriana, Ascidia calliosa, A. paratropa, Pyura haustor, Boltenia villosa, Cnemidocarpa finmarkiensis, and Styela gibbsii


Several Cnemidocarpa finmarkiensis in an old clam
shell. The shell was lying with the tunicates
underneath before the photograph was taken 2X

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Research study 6
 
drawings showing metamorphosis of the larva of the colonial tunicate Botryllus schlosseri into an oozooid and then budding to a blastozooid
The first zooid produced in the colonial tunicate Botryllus schlosseri photograph of a small colony of the colonial tunicate Botryllus schlosserifollowing metamorphosis is known as an oozooid, referring to its origin from sexual reproduction.  Studies at Pacific Grove, California show that the oozooid begins feeding within about 1.5-2d after attachment (at 18-20oC).  After a week or so the oozooid buds and this bud replaces the original.  The new bud and all ensuing buds are known as blastozooids, indicating their origin from asexual reproduction.  Blastozooids are 2-2.8mm long. During growth each blastozooid produces two new buds, then the original is resorbed.   Boyd et al. 1990 Biol Bull 178: 239; photo of B. schlosseri courtesy Stanford Univ marine collection.


Larva of Botryllus metamorphosing into an oozooid. The oozooid soon buds asexually to produce a
blastozooid. The blastozooid then produces 2 buds asexually and is itself resorbed. Growth goes on geometrically in this fashion to form large colonies. The ampullae, that feature so prominently in the
colonial structure of Botryllus, are blood-filled sacs that may be involved in wound repair or defense

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Research study 7
 

Confused? Perhaps this series of drawings will help. They show transformational changes in the colonial tunicate Botryllus schlosseri from tadpole larva through metamorphosis, and then through 4 generations of asexual buddingWatterson 1945 Biol Bull 88: 71.

NOTE  the cycle of growth of new zooids and resorption of old zooids is termed takeover by contemporary researchers

drawing of a 25min larval stage in a series showing events leading from a tadpole larva to a 25d colony in the colonial tunicate Botryllus schlosseri
Note the prominent ampullae and the anteriormost adhesive papulae (the bumps at the anterior end).  The ampullae later become distributed on the periphery of the colony. Siphon locations are shown.
drawing of a 60min larval stage in a series showing events leading from a tadpole larva to a 25d colony in the colonial tunicate Botryllus schlosseri
The larva has attached to the substratum and metamorphosis commences with tail resorption.  The ampullae are more inflated.
drawing of a 7h larval stage in a series showing events leading from a tadpole larva to a 25d colony in the colonial tunicate Botryllus schlosseri
The first member of the colony is termed the oozooid. The ampullae are now scattered around the circumference.  Water flow in this 2-siphoned individual is into the branchial siphon and out the atrial.
drawing of a 2d larval stage in a series showing events leading from a tadpole larva to a 25d colony in the colonial tunicate Botryllus schlosseri
The oozooid forms a bud on the right-hand side, representing the 1st- generation blastozooid.  All subsequent buds are similarly termed blastozooids, indicating their asexual origin.
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drawing of a 4d larval stage in a series showing events leading from a tadpole larva to a 25d colony in the colonial tunicate Botryllus schlosseri
The 1st-generation blastozooid grows in size, and the “mother” oozooid degenerates.  Note the presence of two 2nd-generation blastozoids growing on either side of the 1st- generation one.
drawing of a 5d larval stage in a series showing events leading from a tadpole larva to a 25d colony in the colonial tunicate Botryllus schlosseri
The "mother" oozooid is now nearly resorbed. The 1st-generation blastozooid is growing larger and the two 2nd-generation blastozooids are more prominent.
drawing of a 7d larval stage in a series showing events leading from a tadpole larva to a 25d colony in the colonial tunicate Botryllus schlosseri
The 1st-generation blastozooid has larger 2nd-generation blastozooids.  Note the presence of buds of the 3rd- generation blastozooids (coloured purple) on the 2nd-generation ones.
drawing of a 9d larval stage in a series showing events leading from a tadpole larva to a 25d colony in the colonial tunicate Botryllus schlosseri
The 2nd-generation blastozooids are maturing and the 1st-generation blastozooid is nearly resorbed.  The two 3rd-generation blastozooids are growing larger.
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drawing of a 13d juvenile stage in a series showing events leading from a tadpole larva to a 25d colony in the colonial tunicate Botryllus schlosseri
The 2nd-generation blastozooids are mature and the 3rd generation is developing.  Note the presence of 4th- generation buds on the 3rd- generation blastozooids.
drawing of a 15d juvenile stage in a series showing events leading from a tadpole larva to a 25d colony in the colonial tunicate Botryllus schlosseri
The 3rd-generation blastozooids are nearly mature.  Note that the 2nd- generation blastozooids are nearly resorbed. A common atrial siphon now serves the 4 zooids
drawing of a 17d juvenile stage in a series showing events leading from a tadpole larva to a 25d colony in the colonial tunicate Botryllus schlosseriThe 3rd-generation blastozooids are mature, with 4th-generation buds present, each carrying 5th-generation buds (blue colour). With each new growth phase, or takeover, the number of zooids doubles: growth is thus geometric. drawing of a 25d colony stage in a series showing events leading from a tadpole larva to a 25d colony in the colonial tunicate Botryllus schlosseri
Later (shown here occurring at the 5th generation), the division is asymmetrical, and the 16 zooids split into 2-3 separate systems).  The author comments that the average number of zooids in a system is 8; maximum is 14-15.
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Research study 8
 

drawings comparing fate of the notochord in 4 general of tunicatesTail resorption is by no means standard throughout throughout ascidians.  Of 5 major resorption types described in the literature, 4 are shown here for representative west-coast genera.  The first is illustrated for Distaplia and involves shrinkage and contraction of the epidermis around the tail (contractile tissue is coloured blue in all examples), followed by resorption of the caudal mass.   In the second type, found in Botryllus spp., the tail epidermis is contractile and shrinkage is from the skin-side.  In Boltenia sp. and related species, resorption involves a rupturing of the anterior end of the notochord, and the contents (cells and matrix) flow into the posterior part of the body.  As the tail shrinks the cellular sheath around the notochord becomes compressed and wrinkled.  Finally, in species of Molgula, the cells comprising the notochord contract, and the notochord transforms from a long tubular structure into a short ovoid mass. Cloney 1982 Am Zool 22: 817.

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Research study 9
 
photograph of tunicate Ciona intestinalis
Ciona intestinalis has widescale distribution in Pacific and Atlantic waters. The photo is taken in the UK and the Research Study is done in Australia. Ciona has been introduced widely from Atlantic locations 1.5X

histogram comparing survival of small and large recruits of the tunicate Ciona intestinalisNot surprisingly, adult health and nutritional status have strong bearing on the size and health and, thus, on survivability, graph comparing survival of small and large recruits of the tunicate Ciona intestinalis at different densitiesof offspring in many or all marine invertebrates.  This is especially true in tunicates because the larvae do not feed.  Instead, they rely on yolk material provided by the parent to meet the energy and nutritional requirements of development until they are able to feed for themselves.  Studies on survival of Ciona intestinalis in the field during the first week post-metamorphosis show that large-sized individuals have almost twice the chance of surviving than do small-sized individuals (histogram above).  Density of recruits also has a negative effect on survival, accentuated for smaller-sized individuals (graph on Right).  The authors of the study are unable to explain these density effects on survival.  Marshall & Keough 2003 Mar Ecol Progr Ser 259: 139.

NOTE  sizes are >370µm for the large category and <320µm for the small category.  Larvae are metamorphosed on plastic dishes in the lab and then the dishes are placed in the field for a week in an area where C. intestinalis grows commonly

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Although the authors are unable to provide a definitive explanation for the density effect observed in their week-long study on Ciona intestinalis, they do offer some ideas.  Here is a mix of their good ideas admixed with a few invented not-so-good ones.  Try to identify the latter, then CLICK HERE for explanations.

Individuals overgrow one another at higher densities and the effect is greater for the smaller, more susceptible, individuals. 

More individuals attract more predators and the larger individuals are more likely to survive. 

Small individuals are more likely to be dislodged by currents or by organisms wandering across the plates. 

At higher densities, there is more competition for oxygen in the surrounding seawater. 

At higher densities, food (phytoplankton and other organic matter) becomes limiting. 

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