reproduction, larviculture and early development of the...

Reproduction, larviculture and early development of

the Bluebanded goby, Lythrypnus dalli, an emerging

model organism for studies in evolutionary

developmental biology and sexual plasticity

Sophie Archambeault1, Eric Ng1, Lyle Rapp1, David Cerino2, Bradford Bourque2,

Tessa Solomon-Lane3, Matthew S. Grober4, Andrew Rhyne2 & Karen Crow1

1Department of Biology, San Francisco State University, San Francisco, CA, USA2Department of Biology and Marine Biology, Roger Williams University, Bristol, RI, USA3Neuroscience Institute, Georgia State University, Atlanta, GA, USA4Department of Biology, Georgia State University, Atlanta, GA, USA

Correspondence: S Archambeault, University Of Washington, Molecular and Cellular Biology Program, Box 357275, Seattle, WA

98195-7275, USA. E-mail: [email protected]

Abstract

The Bluebanded goby, Lythrypnus dalli, is a popular

ornamental aquarium species and a key organism

for the study of several fundamental biological

questions, most notably reversible sex change in

adults. To maximize the tractability of this species

as an emerging model system, it is essential to have

an optimized propagation system and a detailed

developmental staging scheme. One limitation to

the larviculture of L. dalli is the relatively small size

of the larvae, which makes the transition from yolk

to feeding challenging. We developed a protocol

and successfully reared three generations of L. dalli

in the laboratory. The protocol contains several key

innovations for the rearing of diminutive fish lar-

vae, including tank design and co-culturing of

microalgae (Isochrysis galbana) with copepods (Par-

vocalanus sp.) in the larval rearing tanks. In

addition, we describe the embryonic and larval

development of L. dalli under controlled conditions

and in comparison with the model organism Danio

rerio. We found that at 21°C L. dalli larvae hatch in

4 days, reach flexion in 18–25 days and are sexu-

ally mature by 3 months. Overall, the embryonic

development of L. dalli is remarkably similar to

D. rerio with several striking differences, including

the position and shape of the blastomere, size of the

neuromasts and corresponding cupula, and relative

timing of pigmentation and brain subdivision. The

ability to rear this species in captivity is a valuable

tool that could be utilized for a variety of similarly

diminutive species and to address a greater breadth

and depth of biological questions.

Keywords: larviculture, co-culture, Lythrypnus

dalli, acanthomorph development, fused pelvic

fins

Introduction

The rearing and development of marine fishes is of

interest to hobbyists, commercial aquarists and

biologists for a variety of reasons including the

following: to increase our understanding of larval

development and comparative embryology,

to increase ichthyoplankton identification, to

strengthen inferences of homology of ontogenetic

characters and to increase the number of species

that can be bred in captivity for ornamental prop-

agation and research (Kendall, Ahlstrom & Moser

1984; Collazo & Bolker 1994). Rearing fish in

captivity provides access to embryos, which is a

limiting factor in evolutionary developmental biol-

ogy research. The ability to rear larvae of various

species in captivity is essential for comparative and

manipulative investigations of the genetic and

developmental basis of diversity and novelty. In

addition, observations of development may help

aquaculturists identify and address significant

hurdles in rearing these and similar species.

Finally, the number of ornamental marine species

© 2015 John Wiley & Sons Ltd 1

Aquaculture Research, 2015, 1–18 doi:10.1111/are.12648

commercially produced in captivity is currently

limited, especially compared to freshwater species

(Andrews 1990; Meirelles, Tsuzuki, Ribeiro, Medei-

ros & Silva 2009; Olivotto, Planas, Simoes, Holt,

Avella & Calado 2011; Sweet 2014), and this rear-

ing protocol may aid in the development of captive

breeding programmes that could potentially allevi-

ate collection pressures on a variety of wild marine

fishes targeted by aquarium trade collections.

The developmental processes of teleost fishes,

with the exception of a few taxa, have not been

well characterized, representing an important gap

in our understanding of animal biology. This defi-

cit is particularly striking as teleosts represent the

majority of vertebrate diversity, with approxi-

mately 27 000 species (Nelson 2006). The most

commonly studied teleost is the zebrafish, Danio re-

rio, a freshwater species widely used in laboratory

research. Accordingly, D. rerio development has

been well characterized (Kimmel, Ballard, Kimmel,

Ullmann & Schilling 1995; Metscher & Ahlberg

1999), which provides a valuable basis for com-

parative developmental studies. However, D. rerio

is not a member of the most speciose and derived

group of teleost fishes, the Acanthomorpha sensu

Near, Eytan, Dornburg., Kuhn, Moore, Davis,

Wainwright, Friedman and Smith (2012), or

spiny-rayed fishes. This group includes many eco-

nomically important species such as tuna, halibut,

rockfish, tilapia and mullet (Stiassny, Wiley, John-

son & Carvalho 2004; Nelson 2006; Eschmeyer &

Fricke 2012). Therefore, building model systems

for developmental studies among acanthomorph

species is of particular interest to both evolution-

ary biologists and aquaculturists.

The Gobiidae are the most diverse family of

Acanthomorpha and the second-most speciose fam-

ily of vertebrates (Moyle & Cech 2004; Nelson

2006). Many are of interest to the aquarium trade,

including the Bluebanded goby, Lythrypnus dalli,

because they are small, brightly coloured and exhi-

bit interesting social and reproductive behaviours

(Sunobe & Nakazono 1993; Scaggiante, Grober,

Lorenzi & Rasotto 2004; Drilling & Grober 2005;

Meirelles et al. 2009). Adult L. dalli are readily col-

lected within their range on shallow reefs using

SCUBA. In addition, adults are readily maintained

in captivity, breed readily and females lay hundreds

of eggs per clutch. Their size at maturity is relatively

small (adult TL less than 6 cm, Miller 1972), mak-

ing them tractable with respect to space limitations

in aquaculture and laboratory settings. The eggs of

L. dalli are attached to the substrate, cared for by

the guarding male (Rodgers, Earley & Grober

2006), and develop into transparent embryos that

are hardy and easily monitored. They are abundant

throughout most of their range, in coastal marine

habitats in the Eastern Pacific from the Gulf of Cali-

fornia to Morro Bay, CA (Miller 1972), and are not

listed as a species of concern in any category (IUCN

Red List, van Van Tassell, Lea & Bearez 2010).

Finally, this species displays several derived traits

that make them ideal for studies of evolution and

development, such as high contrast pigmentation

patterns, pelvic fins that are fused into a sucking

disc (a common feature of this family), and bidirec-

tional sex change. Bidirectional sex change repre-

sents the extreme in sexual plasticity, and L. dalli

are commonly used in studies of sex determination

and sex change (St. Mary 1993, 1994, 1996,

1998, 2000; Black, Reavis & Grober 2004; Black,

Balthazart, Baillien & Grober 2005, 2011; Rodgers,

Drane & Grober 2005; Lorenzi, Earley & Grober

2006, 2012; Rodgers et al. 2006; Rodgers, Earley &

Grober 2007; Lorenzi, Earley, Rodgers, Pepper &

Grober 2008; Lorenzi, Carpenter, Summers, Earley

& Grober 2009; Lorenzi & Grober 2012; Maxfield,

Van Tassell, St Mary, Joyeux & Crow 2012).

Despite their popularity and tractability as a

model for biological enquiry, the early develop-

ment of gobies is relatively poorly studied (Nak-

atsuji, Kitano, Akiyama & Nakatsuji 1997;

Patzner, Van Tassell, Kovacic & Kapoor 2011),

and L. dalli have previously been considered diffi-

cult to rear in captivity (Moe 2008). This difficulty

is largely due to the small size of L. dalli at yolk

depletion and transition to exogenous feeding (L.

dalli larvae measure 2.1 mm at hatching, Patzner

et al. 2011), and the difficulty of providing appro-

priately small live food at first feeding. We devel-

oped a protocol to address this hurdle that can be

adapted to rear a variety of ornamental or

research species with small larvae.

The objectives of this study were to develop a

larval rearing system and protocol for L. dalli, and

to describe their development in an evolutionary

context. We have successfully reared three genera-

tions of L. dalli in captivity and characterized their

embryonic and larval development in comparison

with D. rerio. This comparison highlights the

developmental variation among teleosts and the

need for greater representation of the Acanth-

omorphs for studies that address the bases of novel

traits in a phylogenetic context.

© 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–182

Larviculture and development of the Bluebanded goby S Archambeault et al. Aquaculture Research, 2015, 1–18

Materials and methods

Animals (breeding colonies)

All animal procedures were conducted in accordance

with protocols approved by the Institutional Animal

Care and Use Committees of San Francisco State Uni-

versity (SFSU), Georgia State University, and Roger

Williams University. Adult gobies were collected

using SCUBA from Catalina Island, California and

transferred to SFSU or the Georgia State fish facility.

Embryos were then shipped from Georgia State Uni-

versity to Roger Williams University. Embryonic and

early larval development descriptions were based on

fish housed at SFSU, while the rearing protocol was

developed at Roger Williams University.

Breeding colonies were established with five

adult females housed in 38-litre (10-gallon) rect-

angular tanks with darkened sides. The largest

female in each group usually changed sex within

2 weeks, resulting in operational sex ratios of 1:4

within each colony. Sex change was confirmed by

the presence of fertilized eggs.

Adult tanks were maintained at ambient tem-

perature (~20°C, range = 14.4°C–22.2°C), with

salinity between 30–35, NH3/NH4+ and NO2

<1 ppm, and NO3 <20 ppm. Each tank had iso-

lated, recirculating water filtered with an Emperor

280 BIO-Wheel filter (Marineland Aquarium Prod-

ucts, Moorpark, CA, USA) and received 25%

weekly water changes with filtered, sterilized natu-

ral seawater (Mt. Hope Bay, Rhode Island). Tem-

perature, salinity, flow, fish health and behaviour

were monitored daily and water quality was moni-

tored weekly. A photoperiod of 14:10 hours light:

dark was provided by ambient fluorescent lighting,

mimicking natural conditions during the breeding

season. Tank bottoms were syphoned every day,

or as needed. Adults were fed a variety of food

including Otohime Fish Diet (Marubeni Nisshin

Feed Co., Ltd., Tokyo, Japan), a gelatin based diet

with added seafood products, frozen Mysis shrimp

and frozen bloodworms 3 to 4 times daily until

satiated.

Egg production, incubation and hatching

Each tank contained two 2-inch long, ¾-inchdiameter PVC tubes lined with a plastic transpar-

ency for egg deposition (Fig. 1a). PVC tubes were

visually checked daily for new egg clutches

(Fig. 1b). When eggs were found, the clutches were

inspected, staged and returned to their designated

tubes and tanks for paternal care until hatching,

between 4–6 days later at ~20°C.Eye pigmentation, yolk colour and age of

clutches were monitored to predict hatching. Just

before hatching, clutches were moved to individual

rearing tanks and placed in submerged hatching

chambers (Fig. 1c). Hatching was generally

induced within a few hours after covering larval

tanks to eliminate light penetration. If a partial or

no hatch occurred by the next morning, transpar-

encies were removed from hatching chambers, left

in the air for up to 5 min, shaken mildly, and

replaced.

Larval rearing system

Hatched larvae were reared in 100-L circular

tanks connected to a 380-L recirculating system

(Fig. 1d). The system contained a biofilter and pro-

tein skimmer, and was maintained under the same

temperature and water quality conditions as the

adult broodstock. Rearing tank water levels were

controlled by external T-barbs to maintain the vol-

ume at 75 L. A 40-watt aquarium strip light illu-

minated approximately ⅓ of the tank surfaces,

24 h a day. An 8-cm-diameter cylindrical stand-

pipe screen of 40-lm mesh was attached to the

centre drain of each larval tank (Fig. 1e). A bubble

ring around the bottom of the screen provided

constant gentle aeration and kept larvae and food

away from the standpipe drain. Water inlets were

placed against the sides of the rearing tanks to

minimize turbulence and were fed with constant

flow of 0.1–0.15 L min�1. Daily maintenance

included system water changes of 10–25%, clean-

ing of microalgae drip lines and standpipe screens,

and syphoning of debris and deceased larvae from

rearing tanks.

Plankton Cultures

Larval L. dalli require extremely small foods at the

onset of exogenous feeding. We found that the

copepod nauplii of the genus Parvocalanus sp. were

well suited for the relatively small larvae of L. dalli,

similarly to what has been reported for the lemon-

peel angelfish (Olivotto, Holt, Carnevali & Holt

2006). Parvocalanus crassirostris (Reed Mariculture

Inc., Campbell, CA, USA) were cultured in 150-

litre tanks at densities of ~10–30 copepods per

mL. To maintain these cultures, the microalgae

© 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–18 3

Aquaculture Research, 2015, 1–18 Larviculture and development of the Bluebanded goby S Archambeault et al.

Isochrysis galbana (CCMP 1324) (Florida Aqua

Farms Inc., Dade City, FL, USA) was added twice

daily to densities of 1–2 9 105 cells mL�1, and

water changes of 50% were conducted three times

per week. Copepods were harvested using 40-lmmesh screen, while size-sorted nauplii for larval

feeding were harvested by filtering the culture

through a 75-lm screen filter, then through a 40-

lm screen filter.

Culturing of I. galbana was accomplished follow-

ing the protocols outlined by Creswell (2010). All

pieces of microalgae culture equipment were auto-

claved or sterilized with bleach. Isochrysis galbana

were cultured at ambient temperature (20°C) with

sterilized salt water (salinity of 35) in sealed cul-

ture vessels. Each culture was aerated through a

0.2-lm in-line filter. Optimal densities of

8–12 9 106 cells mL�1 were reached in 5–7 days

in algae media. Cultures were started in 1 L Roux

flasks with autoclaved media, transferred to 20 L

glass carboys and finished in 100 L polycarbonate

tanks (20 and 100 L media was pasteurized to

85°C). Algae media was formulated following the

manufacturer’s instructions (F/2 Algae Food, Fritz

Aquatics, Inc., Texas, USA).

Larval feeding

Copepods (Parvocalanus sp.) were cultured in L.

dalli rearing tanks as described above prior to the

first feeding of larvae. Isochrysis densities were

maintained daily (8–12 9 106 cells mL�1)

through microalgae drip lines as well as direct

addition to the tank. Early larval gobies were able

(a)

(d)

(e)

(b) (c)

Figure 1 Recirculating larval rearing system. (a) Two-inch long segments of ¾ inch PVC pipe were fitted with

transparencies for egg deposition. (b) Bright orange yolks made newly deposited eggs easy to spot within the PVC

tubes. (c) Hatching chambers were 3.8-cm-diameter glass tubes (flower vases) fed with a rigid water line 1 cm from

the bottom, to ensure water movement around the embryos (flow ~ 0.25 L min�1). (d) Design of the larval rearing

system with 380-litre capacity, includes (1) a 150-litre sump containing Kaldnes media for biological filtration, an

in-sump protein skimmer, and a Danner 1800 Mag-Drive pump for recirculation. Plumbing includes (2) recirculat-

ing flow lines fitted with ¼-inch quarter turn labcock valves to control inflow rates, (3) ½-inch PVC return lines

from rearing tanks back to sump, (4) ½-inch PVC alternate waste lines to floor drains, and (5) T-barbs connected to

each centre drain to control the water level in each tank. (e) Customized 8-cm (shown) or 5-cm-diameter centre

standpipe drains kept larvae in the tanks while allowing adequate water exchange. Bubble rings (blue) were fed

from airlines above the larval tanks and kept larvae and copepods away from the standpipe drain.



to consume only the smaller copepod nauplii, and

were therefore reliant on reproducing copepods.

Late stage larvae and early-settled juveniles

were fed Artemia nauplii (Brine Shrimp Direct,

Odgen, UT, USA) in addition to copepod culture

maintenance. Culturing of the copepods through

the addition of microalgae was halted once all the

larvae settled out of the water column. Juveniles

were weaned onto a diet of Otohime pellets

(100–400 lm diameter) at 2 weeks post settle-

ment.

Developmental staging of Lythrypnus dalli

Developmental observations of L. dalli embryos

and larvae were conducted at San Francisco State

University. The individuals used for this study

were housed according to the protocol above,

except that adults were kept in a closed, recirculat-

ing sea water system maintained at 15.5°C(14.4–18.3°C). Developmental observations were

made at two temperatures, 14.4 and 17.0°C.Photos of embryos and larvae were taken at 43

different stages of development from 33 different

clutches of eggs. Embryonic and early larval obser-

vations and photos were taken of live individuals

using a Zeiss DiscoveryV.8 stereomicroscope and

an AxioCam ICc3 camera with AxioVision soft-

ware (Carl Zeiss Microscopy GmbH, Jena, Ger-

many). If necessary, larvae were anaesthetized in

a 1:25 dilution in seawater of a 0.4% MS-222

solution, pH 7 (Tricaine methanesulfonate; Sigma-

Aldrich, Co., St. Louis, MO, USA). Stages of embry-

onic development are described for L. dalli based

on staging criteria for D. rerio (Kimmel et al.

1995) for the following periods: zygote, cleavage,

blastula, gastrula, segmentation, pharyngula,

hatching and early larval. Observations of late lar-

val development were made on individuals pre-

served overnight in 4% paraformaldehyde at 4°C,then transferred to 100% methanol and stored at

�20°C. The proposed staging of late larval L. dalli

was made comparable to D. rerio following Pari-

chy, Elizondo, Mills, Gordon and Engeszer (2009).

In situ hybridization

Whole mount in situ hybridization was done to

test the feasibility of the procedure and to observe

expression patterns of the posterior Hox gene,

hoxa13a, during multiple stages of L. dalli embry-

onic development. Embryos were dechorionated

either before or during overnight preservation in

4% paraformaldehyde at 4°C. Embryos were stored

in 100% methanol at �20°C. Whole mount in situ

hybridizations were carried out as described by

Archambeault, Taylor and Crow (2014).

Results

Fertilization, hatching rates, developmental timing

and survivorship

Fertilization and egg deposition of L. dalli occurred

most frequently in the 4 hours following first light,

8:00–12:00 hours. (Fig. 2). Average nest size at

21.6°C was 835 eggs (n = 9, range 396–1055),

and often represented multiple spawning events.

Males guarded the nests until hatching, and filial

cannibalism was occasionally observed on clutches

that were unfertilized or experienced fungal

growth (personal observations). Removal of

clutches from broodstock tanks significantly

increased the frequency of egg laying: when

clutches were removed, new clutches were fertil-

ized every 9.4 days (SE = 0.86, n = 86) comparedwith every 11.0 days (SE = 0.85, n = 63; Mann–Whitney test: UA=3378, p = 0.0102) when clutchesremained with adults for paternal care. However,hatching rates for L. dalli were more consistent withparental care, ~95% with male care at temperaturesof 18 – 22°C. Survivorship to sub-adulthood wasvariable, between 5–40%.The yolks of L. dalli embryos are orange, making

new clutches easy to detect against the white PVC

tubes. Like other goby eggs, L. dalli eggs are

oblong in shape and are attached to the substrate

by adhesive threads, in contrast to the negatively

buoyant but free moving spherical eggs of D. rerio

(Fig. 3). Tavolga (1950) showed in Bathygobious

that eggs stripped from the ovary are equipped

with the adhesive threads, which are then acti-

vated by contact with water and attach to the

substrate. However, in L. dalli adhesive threads are

absent from the eggs while still in the ovary, and

thus must be added at the time of female egg

deposition (Grober, unpublished data). The female

external genitalia in L. dalli have two internal

chambers, the most ventral of which is most likely

used to produce the adhesive threads during egg

laying (Schuppe et al., in preparation).

Unlike some gobies, L. dalli eggs lack oil globules

(Yokoi & Hosoya 2006; Wittenrich, Turingan &

Creswell 2007). Chorion dimensions are uniform



at 1.88 mm by 0.46 mm (length x = 1.878,

SE = 0.014; width x = 0.464, SE = 0.002; n = 15).Embryonic L. dalli grew in size from 0.57 mm(SE = 0.005) diameter at the zygotic stage to2.72 mm (SE = 0.05) in length at hatching.Lythrypnus dalli embryos hatched in 10.3 days

at 14°C, 9.1 days at 17°C and 4 days at 21.6°C,which is slightly faster than other gobiids reared

at similar temperatures (Fig. 4), but slower than

D. rerio at its common rearing temperature,

28.5°C. To directly compare the development rate

of L. dalli to D. rerio, relative hours of development

was plotted vs. developmental stage (Fig. 4). Both

L. dalli and D. rerio display a linear relationship

between time and developmental stage, thereby

making it possible to estimate developmental stage

based on time and temperature for L. dalli

(y = 0.0189*temp*hpf, R2 = 0.914, R2 = 0.932

for 17°C and 14°C respectively). Interestingly, L.

dalli takes 2.3–3 times longer than D. rerio to

reach the protruding mouth stage, depending on

the temperature. This relationship between

developmental stage and time is robust to temper-

ature, therefore, the following staging scheme for

L. dalli is consistent at a range of developmental

temperatures.

Cleavage, Blastula and Gastrula

The early periods of development are very similar

between D. rerio and L. dalli (Fig. 5a-j), however,

there is an observable difference in blastodisc

position during early cleavage. The cells formed

by early cleavages in D. rerio are positioned on

top of the yolk whereas the cells of the early

blastodisc of L. dalli wrap around the yolk, and

reach towards the vegetal pole (Figs 3b–c, 5a–e).

A third cleavage plane, perpendicular to the ori-

ginal two planes, is established by the 64-cell

stage in both L. dalli and D. rerio embryos, result-

ing in multiple layers of cells (Fig. 5f). By the

seventh cleavage in L. dalli the blastodisc is posi-

tioned on top of the yolk (Fig. 5f). During early

epiboly, the yolk of L. dalli becomes relatively

oblong, mimicking the shape of the chorion

(Fig. 5h–i). By 90%, epiboly the embryo is spheri-

cal again (Fig. 5j).

Segmentation

By the 6-somite stage, both D. rerio and L. dalli

have a distinct head bulge and tail bud, both of

which are more pronounced and relatively bigger

in L. dalli (Fig. 5k). This difference contributes to

an overall oblong shape of L. dalli, mirroring the

shape of the chorion. This difference in shape

appears to be associated with earlier detachment

of the tail and head buds from the yolk in L.

dalli (Table 1). Thus, egg shape may play a role

in shaping developmental progress in some

features.

During the early segmentation period, the optic

placode and somites of L. dalli are often obscured

by a grainy texture, likely due to cell movement

0

10

20

30

40

50

60

12 am–4 am 4 am–8 am 8 am–12 pm 12 pm–4 pm 4 pm–8 pm 8 pm–12 am

Num

ber

of c

lutc

hes f

ertil

ized

Time of day

Lights on at 7 am Lights off at 9 pm

Figure 2 Eggs are fertilized predominantly in the morning. A distribution of the timing of egg fertilization shows

that eggs were most commonly fertilized in the 4 hours following first light. Timing was determined by staging

embryos and estimating their age upon discovery. Age was then subtracted from the time they were found to esti-

mate the time of day that the eggs were fertilized.



(6-somite stage, Fig. 5k). Nonetheless, the optic

placode is visible by the 6-somite stage, the otic

placode is visible by the 9-somite stage, and the

otic vesicle hollows and contains two otoliths by

the 12-somite stage (Fig. 5k–m). Also by the 12-

somite stage, the lens is present and most somites

exhibit the expected chevron-shape.

Differentiation of the brain also occurs earlier

in L. dalli than D. rerio. By the 19-somite stage,

the telencephalon, diencephalon and mesenceph-

alon are evident in the fore- and midbrain of L.

dalli, and are separated from the hindbrain by a

distinct cerebellum (Fig. 5n). In contrast, the

cerebellar primordium does not become promi-

nent in D. rerio until the 26-somite stage

(Table 1).

Pharyngula

The pharyngula period begins with the posterior

advancement of the lateral line primordium over

the trunk somites (e.g. prim-5, prim-15). The pri-

mordium is a difficult trait to monitor, and requires

differential interference contrast optics to visualize.

We took advantage of the large and visible neuro-

masts of L. dalli to characterize their developmental

stage categorically. For example, when a neuro-

mast was visible on the ninth somite, but not yet

on the 14th somite, we categorized the embryo into

the prim 9–14 stage. By the end of the pharyngula

period, neuromasts were visible on the 5, 9, 14, 23

and 29 somites of L. dalli, therefore, we suggest

subdividing the pharyngula period into six stages

(prim 1–5, prim 5–9, prim 9–14, prim 14–23, prim

23–29 and high pec).

Pigmentation begins in the early prim stages

for L. dalli. By prim-9-14, L. dalli has scattered

black and red pigment cells along the ventral line

of the body (Fig. 5o). Additional pigmentation

appears on a dorsal region of somites 14–23 by

the prim 23–29 stage (Fig. 5p). Eye pigmentation

develops with trunk melanocytes in the prim

23–29 stage, which is later than in D. rerio

(Table 1).

Muscle movement, including a visible heart-

beat, begins in L. dalli at the prim 9–14 stage. A

small pectoral fin bud emerges by this stage as

well. The pectoral fin bud develops the apical

ectodermal ridge (AER) and reaches a 1:1 height

to width ratio at the “high-pec” stage (Fig. 5q).

By this stage, many visible changes have

occurred in L. dalli including heavily pigmented

(a)

(c)

(d)

(b)

Figure 3 The eggs of L. dalli are oblong and attached

to the substrate via adhesive threads. (a) The chorions

of 4-day post fertilization L. dalli embryos remain

oblong and attached perpendicularly to the substrate

via adhesive threads (arrows in a and b). (b) The L.

dalli embryo is smaller than the D. rerio embryo (c).

The L. dalli blastomere wraps around the yolk towards

the vegetal pole (marked by arrowheads in b) through

the 8-cell stage. (c) The D. rerio embryo is free-floating,

has a spherical chorion, and the blastomere is perched

on top of the yolk (arrowheads). (d) Ventral aspect of a

cross-section through the body of a female Lythrypnus

dalli, noting the position of the ovaries (Ov), hypaxial

body musculature (M) and genital papilla (GP). The

female GP has two lumens. The dorsal lumen is the

putative oviduct by which the egg is communicated

from the ovary to the genital papilla for adhesion to

the substrate. The ventral lumen is the proposed adhe-

sive thread producing structure within the GP and is

noted with an arrow. Tissue was stained with Haemat-

oxylin and Eosin. Scale bars in b and c equal

0.50 mm, and in d equals 200 lm. Vegetal poles (in b

and c) and ventral surface of the fish (in d) are at the

bottom of the images.



eyes, the appearance of the swim bladder just

posterior to the yolk, and a constriction in the

posterior region of the gut.

Hatching

The beginning of the hatching period is character-

ized by the elongation of the pectoral fin buds

reaching a height to width ratio of 2:1. The fin

buds arch posteriorly, defining the “long-pec”

stage. At this stage, blood circulation is noticeably

strong, and the eyes contain iridescent cells

(Fig. 6a).

The ‘pec-fin’ stage is characterized by the expan-

sion of the apical ectodermal ridge in the pectoral

fins into the paddle shaped apical fold. The blood

6-somite

Protruding mouth

14-somite

Protruding mouth

0.40

0.90

1.40

1.90

2.40

2.90

3.40

3.90

0 25 50 75 100 125 150 175 200

Em

bryo

nic

leng

th (m

m)

Hours post fertilization

L. dalli

D. rerio

64 cell -

18 somite - 25 somite -

high pec - long pec -

pec fin -

prot mouth -

6 somite - germ ring -

75% epiboly -

prim 15 -

prim 25 -

0

10

20

30

40

50

60

70

80

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250

Hou

rs p

ost f

ertil

izat

ion

for

zebr

afis

hat

28.

5°C

Dev

elop

men

tal s

tage

Hours post fertilization

0

5

10

15

20

25

10 15 20 25 30

Mea

n da

ys to

hat

chin

g

Leucopsarion petersii Rhinogobius sp. BI

Elecatinus figaro

Gobiosoma evelynae Zosterissessor ophiocephalus Gobius paganellus

Gobius cobitis

Eviota abax

Eviota storthynx

Priolepis nocturna

Lythrypnus dalli

(a)

(c)

(b)

Figure 4 Lythrypnus dalli embryos are smaller and develop slower than Danio rerio. (a) Embryonic length (EL) as a

function of hpf for L. dalli at 17°C and D. rerio at 28.5°C (D. rerio data taken from Kimmel et al. 1995). Danio rerio is

longer than L. dalli at all stages of development observed, except the 14-somite stage. This increase in EL of L. dalli is

due to the lifting of the tail bud from the yolk. The tail and head buds of L. dalli lift off the yolk sooner than in D. rerio

which results in an increase in EL (6-somite stage in L. dalli and 14-somite stage in D. rerio). This figure highlights the

longer time to hatching of L. dalli compared to D. rerio. (b) Days to hatching as a function of water temperature in 11

species of Gobiidae. Three species were reared at more than one temperature and display an inverse relationship

between temperature and time to hatching. The other eight species were only reared at one temperature, however,

taken together, Gobiidae species show an overall inverse relationship between time to hatching and temperature. Data

for all species except L. dalli were gathered from the literature (Sunobe & Nakazono 1987; Gil et al. 1997; Privileggi

et al. 1997; Arakawa et al. 1999; Borges et al. 2003; Olivotto et al. 2005; Yokoi & Hosoya 2006; Wittenrich et al.

2007; Meirelles et al. 2009). (c) To directly compare the development rate of L. dalli to D. rerio, hours post fertilization

(hpf) were plotted on the vertical right axis against hpf on the horizontal axis for D. rerio (solid black diamonds). The

vertical right axis (hpf) was then translated into developmental stages for D. rerio on the vertical left axis. Developmen-

tal stages of Lythrypnus dalli (vertical left axis) were determined and plotted for various time points (in hpf on the hori-

zontal axis) at 21°C, 17°C and 14°C. Intercepts of the linear trend lines were set to 0, representing fertilization and the

onset of development. Both L. dalli and D. rerio display a linear relationship between time and developmental stage,

thereby making it possible to estimate developmental stage based on time and temperature for L. dalli

(y = 0.0189*temp*hpf, R2 = 0.914, R2 = 0.932 for 17°C and 14°C respectively). L. dalli takes 2.3–3 times longer

than D. rerio to reach the protruding mouth stage, depending on the temperature.



cells have a distinct red pigmentation, and muscle

twitches are strong (Fig. 6b).

The protruding mouth stage is defined by the

extension of the lower jaw anterior to the eyes.

The head and jaws experience major rearrange-

ments, which are evident in the changing shape of

the head (Figs 6a–d, 7). The nares appear just dor-

sal to the upper jaw at this stage. The pectoral fins

have expanded to cover most of the reduced yolk,

and these paddle-like fins are flapped during bouts

of swimming of hatched larvae. The larvae begin

to hatch during the protruding mouth stage in

both L. dalli and D. rerio. Just after hatching, L.

dalli larvae are under 3.0 mm long (x = 2.80 mm,

SE = 0.097).

The most noticeable difference between D. rerio

and L. dalli during the hatching period is that in L.

dalli, the inflation of the swim bladder causes a

noticeable bend in the neural tube. This bend

becomes more prominent during the larval period

(Figs 6c–e, 7).

Post-hatching and Early Larval Period

By the early larval period, significant changes in

the neurocranium and jaw structures result in a

dramatic reshaping of the head. The jaws appear

to gape in most embryos, and the antero-dorsal

slope of the head slants forward to the gaping

mouth (Figs 6e, 7).

One intriguing difference between L. dalli and D.

rerio at this stage of lateral line development is the

relative size of the cupula. The cupula contain kino-

cilia that project distally from hair cells at the base

of each neuromast (Metcalfe, Kimmel & Schabtach

1985). While the neuromasts emerge during the

pharyngula period, the cupula become visible

around 4 days after hatching in D. rerio. In con-

trast, within one day of hatching in most L. dalli lar-

vae, four pairs of cupula visibly protrude from the

trunk lateral line. Up to eight pairs of cupula are vis-

ible along the head and trunk lateral lines of L. dalli

by two days post hatching (Fig. 8). Interestingly,

the cupula of L. dalli are 2–7 times longer than

those of D. rerio, although the larvae themselves are

smaller. Finally, the cupula of L. dalli are visible on

a stereoscope with the correct lighting, making

them significantly easier to observe.

By one-day post hatching, the lower and upper

jaws protrude equally anterior (Fig. 6e). The devel-

opment of the jaws suggests readiness for feeding.

The gape size of L. dalli at hatching is roughly

110 lm tall by 90 lm wide. The yolk is largely

depleted by 4-5 days post hatching at 21°C,at which time food encounters are crucial for

survival.

Larval morphogenesis and settling

Flexion occurs between 18 and 25 days post

hatching, after which caudal fin rays develop, sim-

ilar to D. rerio (Parichy et al. 2009). Mesenchymal

condensations and formation of the fin rays within

the median fin fold quickly follow to form the anal

and second dorsal fins (SL = 3.96 mm, SE = 0.09)

(Fig. 9a).

The paired pelvic buds emerge after the fin rays

have formed in the medial fins including the sec-

ond dorsal, anal and caudal fins (SL = 5.68 mm,

SE = 0.48) (Fig. 9b, e–f). At this stage, several

rows of neuromasts are clearly visible on the cau-

dal fin, which does not occur in D. rerio.

Lythrypnus dalli has two dorsal fins, and the first

dorsal fin emerges from the dorsal midline anterior

to the midline fin soon after the emergence of the

pelvic fin buds (SL = 7.35 mm, SE = 0.11)

(Fig. 9c), similar to what has been described in

Rhinogobius sp. BI (Yokoi & Hosoya 2006). Most

acanthomorphs have two dorsal fins, but the

molecular basis of the differentiated first dorsal fin

has not been studied.

Table 1 Heterochronic developmental characteristics of

Danio rerio and Lythrypnus dalli

Characteristic

Stage

in L. dalli

Stage

in D. rerio

Tail detaches from yolk 9-somite 15-somite

Head detaches from yolk 19-somite Prim-20

Optic placode 6-somite 5-somite

Otic placode 9-somite 14-somite

Otic vesicle with otoliths 12-somite 20-somite

Lens 12-somite 20-somite

Chevron-shaped somites 12-somite 14-somite

Distinct cerebellum 19-somite 26-somite

Eye pigmentation Prim23–29 Prim-15

Trunk melanocytes Prim23–29 Prim-15

Heartbeat Prim9–14 Prim-15

Muscle movement Prim9–14 26-somite

Swim bladder High-pec High-pec

Pectoral fin bud Prim9–14 Prim-25

Iridescent eye pigment Long-pec Long-pec

Visible cupula 1 dph 4 dph

Grey boxes indicate the species in which the trait appears at

an earlier stage.



(a) (b) (c) (d) (e)

(f)

(k)

(o)

(p)

(q)

(l) (m) (n)

(g) (h) (i) (j)



Fin rays are evident in the pelvic fins by SL =7.67 mm (SE = 0.15) (Fig. 9c, g), but the pelvic

fins do not fuse to form the adult sucking disc

until approximately 11.06 mm SL. The fusion of

the pelvic fins occurs in two stages. First, the pos-

terior portions of the two fins fuse together

(Fig. 9g), likely as a result of the outgrowth of the

fins as a single structure at later stages, although

this has not been observed directly. Next, the

anterior edges of the pelvic fins fuse together to

complete the sucking disc structure. A similar pro-

cess is pictured in Rhinogobius sp. BI (Yokoi & Ho-

soya 2006).

Juvenile L. dalli began to settle out of the water col-

umn starting around 40 dph and concurrent with

the onset of adult colouration, after the development

of the pelvic fin sucking disc (Fig. 10), as has been

described for other Gobiidae with pelagic larvae

Figure 5 Early embryogenesis of Lythrypnus dalli. The zygotic (a), cleavage (b–e), blastula (f–g), gastrula (h–j), seg-

mentation (k-n) and pharyngula (o–q) periods. (a) In the 1-cell stage, the blastomere of L. dalli wraps around the

yolk. This continues through the (b) 2-cell and (c) 4-cell stages. Although the cells no longer wrap around the yolk,

they remain in a single layer through the (d) 16-cell and (e) 32-cell stages. Cells are clearly perched on top of the

yolk from (f) the 128-cell stage through (g) the high-cell stage. Cell movement begins during early epiboly. The

layer of cells is uniform in thickness through (h) 50% epiboly. (i) After the formation of the germ ring, there is a

clear asymmetry to the embryo. This asymmetry in cell thickness continues throughout epiboly, including (j) 90%

epiboly. (k) The optic placode is visible by the 6-somite stage, despite the grainy texture of the embryo. (l) The tail

bud lifts off the yolk and the anterior somites are chevron-shaped by the 9-somite stage. (m) The lens and otic pla-

codes are visible by the 12-somite stage. (n) The grainy texture of the embryo is gone by the 19-somite stage, the

head has begun to lift off the yolk, and the cerebellum demarcating the division of the mid- and hind-brain is

evident (arrowhead). (o) Pigmentation appears on the ventral side of the trunk by the prim 9–14 stage. (p) The pec-

toral fin buds emerge (arrowhead) and pigmentation occurs in the eyes and dorsal trunk by the prim23–29 stage.

(q) The pectoral fin buds develop an apical ectodermal ridge, the swim bladder appears and a neuromast develops

on the 29 somite (arrow) by the high-pec stage. All scale bars are 0.50 mm. The first vertical scale bar applies to

a–j. The second vertical scale bar applies to k–n. The horizontal scale bar applies to o–q.

(a)

(b)

(c)

(d)

(e)

Figure 6 Hatching period. (a) By

the long-pec stage, the hindgut

pinches off from the midgut

(arrowhead), the eyes develop

some iridescence, and the ratio of

pectoral fin height to width is

about 2:1. (b) The pectoral fin

takes on a paddle shape by the

pec-fin stage. (c) The lower jaw

protrudes past the anterior edge of

the eye and the neural tube begins

to bend as the swim bladder

inflates (arrow) by the protruding

mouth stage. (d) The mouth gapes

open and most larvae have

hatched by the early larval stage.

(e) Both jaws extend anteriorly,

and the angle of the bend in the

neural tube decreases noticeably in

the larval stage. Scale bar is

0.50 mm and all photos are to

scale.



(Yokoi & Hosoya 2006; Kondo, Maeda, Yamasaki &

Tachihara 2012). There was considerable divergence

in settlement time with some larvae taking

90–100 days to settle. The juvenile period lasted

roughly 1–2 months, at which time most individuals

were sexually mature. This timing is similar to D.

rerio, which reach sexual maturity in 10–12 weeks

(Westerfield 2000).

In situ hybridization

We successfully performed whole mount in situ

hybridizations on embryonic L. dalli and D. rerio

for hoxa13a. The two species display strikingly

similar patterns of expression of hoxa13a in the

trunk, tail bud, gut and pectoral fin buds during

the stages of embryonic development examined

(Fig. 11).

Discussion

Lythrypnus dalli is interesting to aquarists and

evolutionary developmental biologists for its

bright colouration, bidirectional sex change, mod-

ified pelvic fins, size and patterning of neuromasts

and alternative male breeding morphs. In order

to promote research and rearing of this derived

teleost for comparative studies of vertebrate

development and to facilitate large-scale produc-

tion to supply the aquarium trade, an optimized

rearing protocol and developmental staging

scheme were developed. Using this rearing proto-

col, 300–500 juvenile L. dalli can be reared in

each of several tanks at once, allowing for the

production of large numbers of individuals for

research or aquarium use.

Some of the difficulties breeders have faced in

rearing diminuitive species such as L. dalli and

Priolepis nocturna include determining optimal

breeding and rearing temperatures, and culturing

appropriately small foods for onset of first feeding

(Wittenrich et al. 2007). We successfully reared

(a)

(b)

Figure 7 Two different angle measurements proved

helpful in staging L. dalli. a) The head-trunk angle

(HTA), shown in red, and the forehead slope, shown in

blue. (b) The HTA of L. dalli increases in a linear fash-

ion similar to D. rerio, except at an earlier stage. At the

end of the pharyngula period, the jaws of L. dalli pro-

trude anteriorly causing an increase in the slope of the

forehead.

(a)

(b) (c)

Figure 8 The neuromasts and cupula of the head and trunk lateral lines of L. dalli are prominent in recently-

hatched larvae. (a) Eight pairs of neuromasts are marked with dots on a 2 day post-hatching larvae. Three pairs

are located anterior to the pectoral fins on the head lateral line (blue dots), and five pairs are on the posterior lateral

line (red dots). (b) The neuromasts are clearly visible bumps along the posterior lateral line from a dorsal view

(arrowheads). (c) The cupula containing the kinocilia extend at least a body width perpendicular to either side of

the trunk (arrows).



three generations of L. dalli at 18–21°C, near the

top of their reported natural temperature range

(18–22°C, FishBase 2013). We also observed

successful reproduction and development through

early larval stages at a range of temperatures,

from 14°C–22°C, suggesting that this species can

be bred and reared at a range of temperatures.

The relatively small gape size of L. dalli at hatch-

ing and at exogenous feeding have posed a chal-

lenge, as common larval marine fish foods such

as artemia and rotifers (>120 lm) are too large

to consume at first feeding. We therefore devel-

oped and optimized the protocol for the co-cul-

turing of copepod nauplii and L. dalli larvae in

tanks designed to keep the larvae and copepod

nauplii in, while allowing for water and waste

exchange. This protocol is readily adaptable for

rearing other gobies or small marine ornamental

species.

A clear understanding of the evolutionary his-

tory of ontogenetic characters is crucial to the

interpretation of results from developmental

research (Metscher & Ahlberg 1999). To build a

phylogenetic context for multiple ontogenetic char-

acters, developmental studies that include a vari-

ety of vertebrate taxa such as derived teleosts are

essential. Studying development within a phyloge-

netic context identifies interesting developmental

and evolutionary novelties and the appropriate

organisms in which to study them. Our work is

(a) (e)

(b) (f)

(c)

(d)

(g)

Figure 9 Larval development post flexion. (a) Flexion is concurrent with development of the caudal, anal and sec-

ond dorsal fin rays (arrowheads) within mesenchymal condensations of the midline fin. (b) Three rows of neuro-

masts appear on the caudal fin prior to the development of the first dorsal fin. Two pelvic fin buds emerge ventral

to the pectoral fins (arrow). (c) Fin rays appear in the pelvic fin buds (arrow) and the first dorsal fin emerges ante-

rior to the second dorsal fin (arrowhead). (d) The pelvic fin rays elongate posteriorly while the caudal fin rays

branch. (e) Scanning electron micrograph of the ventral anterior side of a larva with emerging pelvic fin buds. Note

the thoracic placement of the pelvic fins ventral to the pectoral fins. (f) Enlargement of the pelvic fin buds in (e).

The pelvic fins emerge as two separate buds that (g) first fuse posteriorly, and later anteriorly (not shown). Photos

are of preserved larvae. Anterior is to the left in all photos. Dorsal is up in a–d, while ventral is up in e–g. Standard

length (SL) of individuals pictured are given for a–d, while average SL for stage pictured are given for f–g. Scale bars

are 1.0 mm. Magnifications are:(e) 144X, (f) 728X and (g) 549X.



the first description of development of a member of

the genus Lythrypnus, and strengthens the phylo-

genetic context for ontogenetic characters within

gobiid species (Sunobe & Nakazono 1987; Gil,

Goncalves, Faria, Almada, Baptista & Carreiro

1997; Privileggi, Ota & Ferrero 1997; Arakawa,

Kanno, Akiyama, Kitano, Nakatsuji & Nakatsuji

1999; Borges, Faria, Gil, Gonc�alves Emanuel & Al-

mada 2003; Olivotto, Zenobi, Rollo, Migliarini, Av-

ella & Carnevali 2005; Yokoi & Hosoya 2006;

Wittenrich et al. 2007; Meirelles et al. 2009;

Kondo et al. 2012). In addition, our ability to

show comparable expression of hoxa13a during

the formation of the gut and trunk in D. rerio and

L. dalli using in situ hybridization suggests that L.

dalli is also tractable for studies of gene expression.

Our comparative analysis has highlighted novel-

ties in L. dalli, such as the blastodisc shape and

large larval cupula. The blastodisc shape of L. dalli

may be due to the oblong shape of the chorion.

However, this unique blastodisc shape has not been

mentioned in other gobiid species, and importantly

not in Priolepis nocturna, a member of a closely

related clade to Lythrypnus (Wittenrich et al.

2007), despite the conserved fusiform shape of the

chorion. Interestingly, the third cleavage in

another gobiid species, Leucopsarion petersii, occurs

in the horizontal plane, which results in two layers

of cells at the 8-cell stage (Arakawa et al. 1999).

In comparison, the third cleavage plane is used

during the sixth cleavage in D. rerio and L. dalli

(Kimmel et al. 1995). These cells are therefore

(a)

(b)

(c)Figure 10 Pigment pattern devel-

opment in L. dalli. (a) Pigmentation

prior to settling is minimal ensur-

ing that larva are relatively trans-

parent. (b) Once the pelvic fins are

well developed and larvae begin to

settle, pigmentation begins to fill.

(c) The adult pigmentation pattern

becomes evident soon after settling

occurs.

(a) (b)

Figure 11 Expression of hoxa13a in the hindgut and tail of Lythrypnus dalli and Danio rerio. Whole mount in situ

hybridization reveals hoxa13a expression in the posterior gut (arrowheads) and tail bud of (a) prim 9–14 stage

L. dalli embryos and (b) prim-10 stage D. rerio embryos. Scale bar is 0.50 mm and all photos are to scale.



perched on top of the yolk, which may be an alter-

native adaptation to the fusiform chorions of the

Gobiidae. Other gobiids may share or have addi-

tional strategies to deal with the size and shape

limitations of their chorions.

Temperature plays a critical role in develop-

mental timing, hatching success and survival of

fish embryos (Schirone & Gross 1968; Kimmel

et al. 1995; Kucharczyk, Luczynski, Kujawa &

Czerkies 1997; Bermudes & Ritar 1999; Arenzon,

Lemos & Bohrer 2002; Yang & Chen 2005; Ahn,

Yamada, Okamura, Horie, Mikawa, Tanaka &

Tsukamoto 2012). Higher temperatures accelerate

development and reduces time to hatching in

many species, including D. rerio (Kimmel et al.

1995). At temperatures outside this range, higher

rates of malformation and lower rates of survival

are common (Kimmel et al. 1995). We observed

shorter times to hatching at higher temperatures

for Lythrypnus dalli in the range tested (14.4°C–21.6°C), and did not observe higher rates of mal-

formation or death, suggesting that L. dalli devel-

opment progresses normally within this

temperature range. Danio rerio occur in warmer

waters than L. dalli, and therefore are kept in

warmer water, develop faster and hatch sooner

than L. dalli in the lab.

Interestingly, despite the temperature and over-

all time to hatching differences in L. dalli and D.

rerio, we found that both species progress through

larval and juvenile stages in a linear fashion.

While neither species allocates more time to any

particular developmental stage based on the stag-

ing criteria proposed here, and following Kimmel

et al. (1995), there were several variations in the

timing of specific characters, which appeared at

different developmental stages between the taxa

(Table 1). For example, the otic placode, otoliths

and lens develop at earlier developmental stages in

L. dalli, and eye pigmentation occurs earlier in D.

rerio, indicating variation in timing of individual

organs with respect to developmental stage. Inter-

estingly, some characteristics of the segmentation

period develop at similar stages in the ice goby,

but at later stages in D. rerio (Kimmel et al. 1995;

Arakawa et al. 1999), which suggests an evolu-

tionary component to these differences in develop-

mental timing. In addition, L. dalli are smaller

than D. rerio at all embryonic stages and times,

with the exception of the 14-somite stage, likely

due to the lifting of the head and tail bud off the

yolk at an earlier stage, which appears to be a

shared feature of gobiid embryos, again perhaps

associated with the shape of the chorion.

The pelvic fins of L. dalli are of interest for sev-

eral reasons. First, they articulate in the thoracic

region as is common in many acanthomorphs, rel-

ative to the abdominal pelvic fins of D. rerio. Sec-

ond, the pelvic fins of L. dalli are fused on both the

posterior and anterior edges, as are the basiptery-

gia, the internal bony pelvic girdle supporting the

pelvic fins. These modifications to the pelvic fins

form a sucking disc – a unique morphological

character of many gobies – in a similar manner as

described in Rhinogobius sp. BI (Yokoi & Hosoya

2006). These pelvic fin modifications make mem-

bers of the Gobiidae, including L. dalli, interesting

taxa in which to study the evolution of fin modifi-

cations and body plan diversity.

An interesting developmental trait of L. dalli is

the pronounced neuromasts and cupula of the

hatching-stage embryos and early larvae. We

found eight pairs of neuromasts and corresponding

cupula on newly hatched larvae, which extended

up to a body width out of the neuromast. Compar-

atively, the neuromasts of D. rerio are much smal-

ler and difficult to find. Similarly pronounced, and

comparable numbers of cupula have been reported

in two Eviota species, E. abax and E. storthynx

(Sunobe & Nakazono 1987), but were specifically

not found in a more closely related species to Lyt-

hrypnus, Priolepis nocturna (Wittenrich et al. 2007).

We found no other reports of these cupula in lar-

val gobiids, however, preservation in 5% formalin

may destroy them (Sunobe & Nakazono 1987). It

would be interesting to further examine the preva-

lence of these large cupula and neuromasts in gob-

iid species as well as in a variety of pelagic and

demersal larval fishes.

Several distinct lineages within the Gobiidae

family are bidirectional sex changers (St. Mary

1993; Sunobe & Nakazono 1993). This is a rare

characteristic in teleosts and represents an extreme

in sexual plasticity. Little is known about the

development of the gonads of these species, but

the limited data suggest similar molecular path-

ways are involved in initial gonad development

and the reallocation process of gonadal tissue dur-

ing sex change (Miyake, Sakai & Kuniyoshi 2012).

In L. dalli, preliminary data indicate that juveniles

develop initially into a bipotential phase where

gonads contain both eggs and sperm and genitalia

are ambiguous (Lorenzi and Grober, unpublished

data), followed by social regulation of initial sexual



development that is responsive to social status

(Solomon-Lane, et al., in preparation) in the same

way that has been shown for adults (Rodgers et al.

2007).

Lythrypnus dalli is a small ornamental fish

that is easily maintained in a laboratory setting.

Furthermore, adults readily lay eggs in captivity,

and are of interest for studies of sexual plasticity,

sex change and alternative reproductive strategies.

This taxon is tractable and evolutionarily interest-

ing for studies of developmental traits and novel-

ties, such as the early neuromasts of the lateral

line and modified pelvic fins. This work provides a

useful staging scheme that can be used to study

the development of several novel features in a

representative derived teleost.

Acknowledgments

We thank Darren Gewant for his support and

expertise. Funding for this research was provided

by the National Science Foundation (proposal num-

ber 1022509 and grant number DUE-0728279

awarded to Dr Karen Crow and San Francisco State

University, respectively; # IOB0548567 to Mat-

thew Grober and a Doctoral Dissertation Improve-

ment Grant (DDIG) #1311303 to Tessa Solomon-

Lane) and a Sigma Xi Grant-in-Aid of Research to

Tessa Solomon-Lane. Funding was also provided by

the Institute of Museums and Library Sciences for

the work carried out at Roger Williams University.

The authors declare no competing interests.

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