UNIVERSITY OF HAWAI'I LIBRARY

REPRODUCTIVE BIOLOGY OF SANDWICENSIS, A HAWAIIAN STREAM GOBIOID

A THESIS SUBMlITED TO THE GRADUATE DMSION OF THE UNIVERSITY OF HAWAI'I IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

IN

ZOOLOGY (ECOLOGY, EVOLUTION AND CONSERVATION BIOLOGy)

DECEMBER 2006

By TaraK. Sim

Thesis Committee:

Robert Kinzie, Chairperson Kathleen Cole Michael Kido We certify that we have read this thesis and that, in our opinion, it is satisfactory in scope and quality as a thesis for the degree of Master of Science in Zoology.

THESIS COMMITTEE

i Acknowledgements

Committee members: R Kinzie, K. Cole, M. Kido. P. Ha for guidance and encouragement. J. Efird for statistical analysis. T. Carvablo, R Shimojo for assistance with laboratory techniques. Q. He for histology help. G. Arakaki and H. Sim for help in the field. S. Togashi for technical support and field assistance. Limahuli National

Tropical Botanical Garden and Hale. Support for this project was provided by EPSCoR

ii Abstract

Spawning season, size at first reproduction, oocyte maturation, fecundity and spawning frequency ofEleotris sandwicensis, an amphidromous Hawaiian gobioid, were studied from July 2004 through December 2005 in Nuuanu stream, Oahn, Hawaii. The smallest male and female fish with mature gonads measured 54 mm standard length.

Ripe individuals were collected in all months, and gonadosomatic index was highest in males and females from June 2004 through February 2005. Size-frequency distributions of measurements of vitello genic oocyte diameters and microscopic observations of oocytes indicated this species has asynchronous oocyte development. Estimates of batch fecundity ranged from 5000 eggs to 55000 eggs. Batch fecundity was positively related to standard length, wet weight, and ovary weight, and inversely related to oocyte diameter. appears to be capable of repeat spawning.

iii Table of Contents

Acknowledgements ...... il

Abstract ...... •...... iii

List of tables ...... v

List of figures ...... •...... vi

Introduction ...... •.•...... 1 Studies on Hawaiian stream gobioids ...... •...... 1 Study organism ...... 2 Amphidromy ...... •.•.•...... 3

MaterialsIMethods ...... •..•....•.•...... 3 Nuuanu Stream study...... ••.•....•...... 3 Work on additional streams ...... •...7

Results ...... •...... 7 Nuuanu Stream study ...... 7 Work on additional streams .••••.•.•.•.•••.•.•••••••••••••••••••••••••••••••.•.•.••••. 11

Discussion ...... 11

Appendix ...... •...... 30 Appendix 1: size frequency histograms ...... 30 Appendix 2: female data .••••.•.•.••...... •.•.....•••.••••....•.•.•.....•...... 35 Appendix 3: male data ...... •.•.•....•.•.•...••.•...... ••...•....•.•37

References ...... •..•...... 39

iv List oftables

Table ~

1. Specimen collection data for Nuuanu ...... 18

2. Oocyte stages in Eleotris sandwtcensis .•...... 19

3. Collection data from 6 streams (males) ...... 20

4. Collection data from 6 streams (females} ...... 21

v List of figures

Figure ~

1 Oocyte stages ofE. sandwicensis ...... •...... 22

2 Testes stages ofE. sandwicensis ...... 23

3 Size at maturity of females ...... 24

4 Size at maturity of males ...... 25

5 Spawning season of females ...... 26

6 Spawning season of males ...... 27

7 Standard length comparisons ...... 28

8 Wet weight comparisons ...... •...... 29

vi Introduction

Streams provide habitat to Hawaii's native freshwater macrofauna, which includes five gobioid , three mollusks and seven decapod crustaceans (Timbol and

Maciolek 1978). Of the five species of gobioid fish native to Hawaiian freshwater systems, 4 species are true gobies, belonging to the family Gobiidae (Timbol and

Maciolek 1978). The other, Eleotris sandwicensis, is a sleeper belonging to the family

Eleotridae (Gosline and Brock 1960). Eleotrids are common inhabitants of tropical coastal habitats throughout the world (Winemiller and Ponwith 1998). With gobies, they are important components of stream and estuarine systems on tropical oceanic islands

(Fitzsimons, et al1996; Winemiller and Ponwith 1998). The family is composed of35 genera and roughly 155 species (Nelson 2006). Distribution ofeleotrids extends as far north as the Atlantic coast of the United States and as far south as Stewart

Island, New Zealand (Nelson 2oo6). Historically, Eleotris sandwicensis was an important cultural resource to Native Hawaiians, being one of the favorite food fishes

(Titcomb 1978).

Studies on Hawaiian stream gobioids

Although little work has been done on the biology of Eleotris sandwicensis, the reproductive biology of other Hawaiian freshwater gobioid species have been studied.

Kinzie (1993) studied the goby Lentipes con color, and reported the breeding season from

October to June, with a peak in late winter and early spring. Females are believed to

spawn more than a single clutch per breeding season (Kinzie 1993). Ego (1956) and Ha

and Kinzie (1996) studied Awaous guamensis and found it spawns between August and

1 December. In A. guamensis, size at first maturity was determined to be 73 mm standard length in both males and females. Fecundity was estimated at 117,600-689,500 eggs per ripe female, and females produce only a single clutch per season (Ha and Kinzie 1996).

Tomihama (1972) studied and found that spawning occurs between

August and March, however no fecundity data were given for this species.

Today, a number offactors threaten the continued existence of Hawaiian stream species (Timbol and Maciolek 1978; Hathaway 1978; Brasher 2003). As Hawaii became increasingly urbanized, many streams underwent dramatic physical and biological alterations (Maciolek 1977; Hathaway 1978; Way, et al. 1998). A survey done by the US

Fish and Wildlife Service between 1975-1976 found that 15% of Hawaii's perennial streams had been channelized and that 53% had water diversions (Timbol and Maciolek

1978). The survey also showed that species diversity and numbers of native stream were lower in altered streams than in unaltered streams (Hathaway 1978; Norton et al. 1978), and all streams sampled contained exotic species (Timbol and Maciolek

1978).

Study organism

Eleotris sandwicensis is endemic to the Hawaiian Islands, and is commonly found in the estuaries and lower reaches of mountain streams (Fitzsimons et al2002). It can be found on all the main Hawaiian Islands, and is especially abundant on Oahu (Yamamoto and Tagawa 2000). It is thought that the species is carnivorous, feeding on a wide variety of foods including other fishes (including other E. sandwicensis), crustaceans, and aquatic gastropods (Fitzsimons, et al 2002).

2 Amphidromy

Eleotris sandwicensis, like the other four native Hawaiian gobioids, is amphidromous (McDowall 1987). Amphidromy is a type of diadromy in which fish migrate between freshwater and marine environments (McDowall 2003). Demersal eggs of E. sandwicensis are oviposited and fertilized in the stream. Newly hatched larvae are swept out to sea, where they develop for up to 160 days in the oceanic plankton before recruiting back to freshwater where they will complete the rest of their life cycle (Radtke et al. 1988; Lindstrom 1999). Ancestors of native Hawaiian gobioids likely colonized

Hawaiian streams as larvae of amphidromous species accidentally carried by ocean currents from streams elsewhere in the Indo-Pacific (Watson 1991, 1992; McDowall

2003). Evidence from genetic studies on the four gobiid species does not support the idea of homing to natal streams in any of the species, thus there is no evidence for genetic structuring of discrete island populations (Chubb et al. 1998).

Materials and methods

Collections

Male and female Eleotris sandwicensis were collected from Nuuanu Stream on the island of Oahu. Collections were made at the lower reach of this stream, under the H­

I freeway overpass. Nuuanu stream is a continuous stream that is modified by having concrete lined channels, alien vegetation, channel realignments, elevated culverts, revetments, and blocked or filled-in channels (Timbol and Maciolek 1978). Non-native organisms present include poeciliids, cichlids, small-mouth bass (Micropterus

3 doiomieui), and armored catfish (Hypostomus c.f. watwata) (Yamamoto and Tagawa

2000). This stream runs through urban Honolulu passing under the H-1 freeway and is considered degraded due to the degree of physical and biological alterations it has

undergone.

Fish were collected monthly from Nuuanu stream (21°19'19"N; 157°51 '36''W) between July 2004 and December 2005, for a total of 17 months. No fish were collected

in November 2004 due to heavy flooding ofNuuanu stream. Fish were collected during

the day between 13:00 and 15:00 using size 16 limerick hooks baited with shrimp. After collection, fish were sexed by visual examination of the urogenital papillae. Standard

and total length were recorded and fish were euthanized in an overdose ofMS222. Fish were then labeled and fixed in 10% buffered formalin. Fixed fish were weighed to the nearest O.OOOlg with a digital scale, then ovaries and testes were removed. Each ovarian

and testicular lobe was weighed separately to the nearest 0.0001 g and stored in 10%

buffered formalin. Somatic weight was calculated by subtracting gonad weight from total body weight.

Gonad maturation staging

Ovaries of each female were staged based on size and appearance of whole

preserved oocytes observed using wet-mount preparation under a light microscope at 40x

magnification. Testes were staged using histology. Gonads from 24 females and 17

males in various stages of maturity were histologically examined for closer analysis of

maturation stages and for validation of wet-mount oocyte staging. Gonads were

embedded in Paraplast plus brand paraffin and cut into sections of 7pm thickness with an

American Optical microtome. Sections were placed on microscope slides and stained

4 with Mayer's hematoxylin and Eosin Y. Coverslips were mounted on sections using pennount mounting medium. Stained sections were then observed at 40x magnification.

Sections of ovaries were compared to observations of oocytes in various maturation stages made using wet mount preparation. The purpose of histologically preparing the oocytes was to validate the accuracy of staging by wet-mount observation (Ha 1991).

Maturation stages of oocytes were determined following Wallace and Selman (1981) and

Cole (2002). Using an ocular micrometer, oocyte size-frequency distributions were determined for each oocyte maturation stage by measuring the diameters of oocytes that had been sectioned through the nucleus.

Spawning season and size at maturation

Spawning season and size at maturation of males and females were estimated using the gonadosomatic index (GSl) of all fish collected. GSI is calculated as follows:

(gonad wet weight)/(somatic wet weight) x 100. It is the percentage of gonad to somatic body weight and is a common way to quantitatively denote reproductive effort in fish

(Miller 1984). Spawning season was estimated for males and females collected from

Nuuanu stream by plotting the GSI of each fish collected versus the month of collection

(Ha 1991; Texeira 1994; Winemiller and Ponwith 1998; Privatera 2002). Size at maturity was estimated for males and females collected from Nuuanu stream by plotting GSI versus standard length for each fish collected (Ha 1991). The smallest male and female with mature gonads, as indicated by GSI and histology, were determined from the plot.

Fecundity

A volumetric method was used to estimate batch fecundity for the species, which is defined as the number of vitellogenic eggs in the ovary just prior to spawning (Bagena1

5 and Braum 1971). Ovaries offemales with elevated GSI collected during the spawning season and proven to be ripe through histology were used to estimate fecundity. Right and left ovarian lobes were weighed separately. A single lobe from each female was teased apart and the freed oocytes were placed in a vial with a known volume of distilled water. The vial was shaken 10 times to get the oocytes into suspension, and 1 ml of the suspension was immediately removed with a volumetric pipette. Oocytes in the 1 ml aliquot were placed onto an egg-counting slide, and all late stage vitellogenic oocytes were counted under a compound microscope. Counts were recorded, and the oocytes were returned to the suspension. Five 1 ml replicates were sampled from the suspension with, and the average of the 5 replicates was taken. This number was then used to estimate the number of late-stage vitellogenic oocytes in the ovarian lobe (the total number oflate stage oocytes in the entire suspension). To estimate total batch fecundity of each female, the weight of the ovarian lobe from which oocytes were counted and the weight of the other ovarian lobe were used to extrapolate the number oflate stage vitellogenic oocytes in the uncounted lobe. Fecundity of both lobes was then added to obtain an estimate of total batch fecundity.

Spawningfrequency offemales

The diameters of 100 randomly selected vitellogenic oocytes from 17 ripe females were measured using an ocular micrometer. The data were then placed into a size­ frequency histogram to examine whether or not there was a bi-modal distribution of oocyte diameters within individuals. Ifthe species is capable of repeat spawning, the oocyte size frequency distribution should be at least bi-modal, reflecting more than one modal group of vitellogenic oocytes within the ovary, including an advanced group that

6 will provide the next spawning, and a group of ripening oocytes that will be spawned

after the first group (Rogers 1989; Fonda 1993; Privatera 2002).

Work on additional streams

Collections of Eleotris sandwicensis were made from five additional streams on two additional islands. The purpose was to compare data from the population in Nuuanu

stream, a degraded stream, with populations in other streams of varying quality.

Collections were made from the following 'degraded' streams: Hanamaulu stream on

Kauai, and Wailoa River on Hawaii Island. Collections were made from the following

'pristine' streams: Kahana stream on Oahu, Limahuli stream on Kauai, and Hakalau

stream on Hawaii Island.

Results

Specimen collection

Fish were collected from Nuuanu stream between July 2004 and December 2005.

Data for all fish collected from Nuuanu stream are in Table 1.

Oocyte maturation staging

Following criteria used by Wallace and Selman (1981) and Cole (2002),5 oocyte

maturation stages were identified in female Eleotris sandwtcensis. These stages are

described in Table 2 and Figure 1.

The ovaries of three females contained oocytes at all five stages of maturation.

The majority of the ovaries examined had Stage I, IT, m, and IV oocytes in the ovary at

onetime.

7 Maturation stages oftestes

Three maturation stages were identified in E. sandwicensis testes following Ha

(1991). Stage I (immature) testes contained only primary spermatocytes as evidenced by closely spaced, strongly staining nuclei. There were no free sperm present in the testes lobules. Stage n and stage ill testes were spermiated, and were differentiated by the amount of sperm they contained. Stage II (intermediate) testes had only a few free sperm in the testes lobules. Stage ill (mature) testes had free sperm packing the testes lobules.

These stages are illustrated in Figure 2.

Critical GS!for maturity

A critical threshold OSI for maturity was estimated to be 0.67 in females. This is because no female with a OSI below 0.67 had vitellogenic oocytes in the ovary (N=6), as determined by histology and wet mount observations of samples of ovaries. No female with a OSI above 0.67 had immature ovaries (N=4S), defined as ovaries in which there are no vitellogenic oocytes present, as determined by histology and/or wet mount observations of oocytes. In males, the critical OSI for maturity was determined to be

0.204. A male was determined to be mature if free sperm were present in the testes lobules (Munday et al. 2002). All males collected with OSI below 0.204 (N=6) had immature testes, as demonstrated by the absence of free sperm in the testes lobules when examined histologically. Some of the histologically examined males with OSI above

0.204 had mature testes (N=4), although some had immature testes based on histological

8 examination (N=4). GSI was not positively correlated with female SL (p= 0.874;

1"=5.2%) or male SL (p=O.647; 1"=6.6%).

Size at maturation

The smallest female collected with mature gonads was 54mm SL. This fish was collected in October 2005 and had a GSI of3.017 (Figure 3). The smallest male collected with mature gonads was 54mm SL. This fish was collected in January 2005 and had a GSI of 0.363 (Figure 4).

Spawning season

Females with mature gonads were collected in all months of collection (Figure 5),

suggesting year-round reproduction in Nuuanu stream. Mean female GSI was lowest in

March, April, and May 2005. GSI was highest from June through November 2005.

Individuals with high GSI, defined as being higher greater than the critical GSI for maturity, were collected throughout July 2004 to January 2005 (Figure 5).

Males with mature gonads were collected in all months (Figure 6), suggesting

year-round reproduction. Mean male GSI was lowest in April and May 2005. GSI was

highest from June through November 2005. In December 2005, male GSI dropped

slightly. However, all males collected in December 2005 had a GSI above the critical

GSI for maturity. Individuals with a high GSI relative to the critical GSI for maturity

were collected throughout July 2004 to March 2005 (Figure 6).

Fecundity

9 Fecundity was estimated for 18 females from Nuuanu stream with GSI ranging

from 2.S20 to 8.110. Estimates of batch fecundity ranged from 49S0 eggs for one S4 mm

SL female with GSI 3.26, to S4760 eggs for one 109 mm SL female with GSI S.12.

Regression analyses were done on fecundity versus the following variables: SL

(fecundity = -42322 + 716(SL); r"=S3.9%; P = 0.001) and wet weight (fecundity = -S6S3

+ I 72S(wet weight); r"=73.0%; p = 0.00). Fecundity was positively correlated with both

of these variables, and more strongly correlated with wet weight.

Oocyte diameters Oocyte diameters were measured from 17 of the females used in fecundity

analysis. Size frequency histograms are in appendix I. Regression analyses were done

on mean oocyte diameter versus SL (mean oocyte diameter = 0.307 - 0.000020(SL);

r"=O.OO%; p = 0.973); and wet weight (mean oocyte diameter = 0.303 + O.OOOIS(ww);

r"=O.OO%; p = 0.892). Neither SL nor wet weight were significant predictors of mean

oocyte diameter.

Spawningfrequency offemales

Oocyte size-frequency histograms were constructed for 17 female E. sandwicensis

collected from Nuuanu stream between the months of July 2004 and February 200S and

the months of June 200S and November 200S. The majority of these females showed a

single clutch developing in the ovary at one time. However three females showed a small

batch of smaller-diameter oocytes somewhat separated from the main batch. It is

possible that these represent a batch of oocytes that is still ripening and will be ovulated

after the first batch. An 82mm SL female weighing 11.0Sg was collected in September

200S. An 89mm SL female weighing 17.49g and a 70mm SL female weighing 7.23g

were collected in November 200S. Because each of these fish was collected in what was

10 probably the middle of the spawning season, it is possible that these fish would spawn another batch after spawning the first batch of vitellogenic oocytes.

Work on additional streams

Fish were collected from Kahana stream (Oahu), Wailoa stream and Hakalau stream (Hawaii Island), and Limahuli stream and Hanamaulu stream (Kauai). Data from these collections are in Tables 3 and 4. Figures 7 and 8 depict the relationships among

fish from these 6 streams in terms of standard length and wet weight. ANOV A results

indicate that male and female E. sandwicensis collected from high quality streams were

significantly larger than fish collected from low quality streams, both in terms of standard

length and wet weight (p<0.0001). For females, both island and stream condition have

significant effects on standard length (ANOV A, both p< 0.0001) but no significant

interaction effect (p = 0.57). For males, both island and stream condition have significant

effects on standard length (ANOVA, both p< 0.0001) but no significant interaction effect

(p = 0.06). For female wet weight, both island and stream condition have significant

effects (ANOV A, both p< 0.0001) but no significant interaction effect (p = 0.10). For male wet weight, both island and stream condition have significant effects (ANOV A, both p< 0.0001), and also a significant interaction effect (p = 0.01).

Discussion

Oocyte maturation dynamics

Oocyte development in Eleotris sandwicensis is asynchronous. According to

Wallace and Selman (1981), the asynchronous ovary consists of oocytes at all stages of

development, and is typically found in species which spawn many times during a

breeding season, as has been reported for most gobioid species (Miller 1984). Further

11 evidence of an asynchronous type ovary is the presence of stage I (chromatin nucleolar) oocytes in alI of the ovaries that were histologically examined. The presence of oocytes at this stage indicates asynchronous oocyte development because it is likely that these small young oocytes are constantly being recruited into the maturing stages in continuous maturation of the oocytes (Arocha 2002).

Testes development

Histological examination shows that Eleotris sandwicensis has an unrestricted spermatogonial testis type, as described by Grier (1981). In this type of testis, spermatogonia may be distributed along the entire length of the testis lobules. This type oftestis is typical of most teleosts. OSI was not a good indicator of maturity in E. sandwicensis males. A few males with relatively high OS! had testes with no free sperm, while some males with low OSI had mature testes with free sperm packing the testes lobules. Thus, caution should be exercised when attempting to determine male maturity and spawning activity based on OSI alone. A more reliable approach would be to use histological examination of the testes. Regression analysis of OS I vs. SL showed that there was no relationship between OSI and male standard length. Thus, larger males do not necessarily have higher OS! than smaller males. This could indicate the use of alternative mating tactics in this species. Various studies on gobiids have shown that these fish commonly employ alternative mating strategies (Magnhagen 1998; Mazzoldi et al. 2000; Rasotto and Mazzoldi 2002). The most common alternative mating strategy is one involving nest-holding males who attract females using dominance or courtship, and sneaker males who 'parasitize' the reproductive efforts of nest-holding males by fertilizing eggs in the nests of these males (Taborsky 1994; Gross 1996; Henson and

12 Wamer 1997). Studies have shown that smaller male grass gobies (Zosterisessor ophiocephalus) had higher OSI than larger males, indicating the presence of alternative mating tactics (Mazzoldi et al. 2000). This is because sneaker males have larger testes which release more and faster diffusing sperm which can quickly fertilize eggs (Rasotto and Mazzoldi 2002). Because these smaller males do not invest in nest guarding or parental care as the larger males do, they can instead invest energy into sperm production

(Mazzoldi et al. 2000).

It is not possible at this time to say whether male Eleotris sandwicensis in Nuuanu stream employ an alternative mating strategy. The lack of correlation between SL and

OSI and the bias towards small individuals suggests that this may be the situation in

Nuuanu stream. The presence of many small males with high OSIs could indicate that a sneaking strategy might be present in this population, but without observations of nesting behavior, it is not possible to be sure of this.

Size at maturity

The smallest male and female fish collected with mature gonads measured 54mm

SL. Both fish had OSI well above the threshold OSI for maturity. However, these were the smallest male and female specimens collected overall, regardless of OSI. Thus, it is not possible to say that 54 rom is the minimum length fish must attain in order to become mature. It is only possible to say that a male fish and a female fish as small as 54mm are capable of attaining maturity. To accurately determine the minimum size Eleotris sandwicensis must obtain to reach maturity, it would be necessary to collect more specimens in this size and smaller classes. The conspicuous absence of very small individuals in collections may indicate that hook and line is not a suitable method for

13 collecting these small specimens. A less selective method such as electrofishing may be necessary to collect individuals in this size class.

Spawning season

The presence of females with mature ovaries in all months of collection suggests the potential for year-round reproduction by female Eleotris sandwicensis in Nuuanu stream. GSI was lowest in the spring months. However, individuals with mature ovaries were still present in collections for these months. From late summer through winter, GSI of females remained relatively high. Males with mature testes were collected in all months. There appears to be a drop in male GSI during spring, corresponding to that of females. These data suggest that spawning activity of E. sandwicensis in Nuuanu stream is reduced during March. April, May, and June.

Studies on other tropical eleotrids have found evidence of year round spawning.

Winemiller and Ponwith (1998) studied the eleotrids Eleotris amblyopsis, Dormitator maculatus, Eleotris pisonis, and Gobiomoros dormitor in Costa Rica and found evidence of year-round reproduction in all four species. Coates (1992) found evidence of year­ round reproduction in the eleotrids Oxyeleotris heterodon and Ophieleotris aproros.

Studies on other species of Hawaiian stream gobioids have indicated that their spawning seasons coincide with the Hawaiian rainy season (Tomihama 1972; Ha 1991;

Kinzie 1993; Lindstrom and Brown 1994). Spawning during periods of increased discharge could be beneficial to these species because it may increase the probability that larvae will be swept from the stream to the ocean where they will be able to continue development Data from Nuuanu Stream suggest that E. sandwicensis continues to spawn during the dry season of the year, defined as April through October (Pukui and

14 Elbert 1986), although spawning in the months of March 2005 through May 2005 is likely depressed in both males females, as evidenced by relatively low GSIs. It appears that E. sandwicensis has an extended breeding season, as ripe individuals were collected in all months of the year.

Fecundity

Regression analysis shows that batch fecundity is positively correlated to female standard length and wet weight. In these relationships, p-values were significant, indicating that both factors are significant predictors of fecundity in Eleotris sandwicensis. Fecundity can be more accurately estimated by female wet weight, which had the higher fl. Oocyte diameters were not significantly predicted by either SL or wet weight.

Spawningfrequency

Only three female E. sandwicensis had histograms that were bi-modal. While this might suggest that the species spawns only a single batch of oocytes per season, histological examination of the ovaries supports repeat spawning. The presence of oocytes in different stages of maturation is indicative of repeat spawning, and suggests there is continuous recruitment from the batch of chromatin nucleolar oocytes within the ovary (Arocha 2002). This is in turn indicative of the asynchronous type ovary typical of iteroparous species (Wallace and Selman 1981).

Work on additional streams

Plots comparing standard length and wet weight (Figures 7 and 8) show that fish from 'pristine' streams are typically longer and heavier than fish from 'degraded' streams. When fish from Nuuanu stream are compared to fish from the three 'pristine'

15 streams, it is clear that Nuuanu fish were smaller, both in texms oflength and weight.

Data suggest that Eleotris sandwicensis in Nuuanu stream are not representative of the species as a whole. The most marked difference in size between two populations is that between the Nuuanu Stream fish and the Limahuli Stream fish. Fish collected from

Limahuli Stream (pristine) were much larger than fish collected from Nuuanu Stream; however, the majority of the Limahuli Stream fish had immature gonads whereas the majority of the Nuuanu fish were mature. Data thus suggest that E. sandwicensis from

Nuuanu Stream stream mature at a smaller size, compared to E. sandwicensis, in

Limahuli Stream.

Size differences and states of maturity in these different populations offish should be of special interest. It may be that size at maturity is larger in Limahuli Stream fish, which can have many advantages in texms of reproductive success. The absence of non­ native predators on adults likely plays a large role in this. In I jmahuli Stream, E. sandwicensis is the top predator. With the exception of the Tabitian prawn,

Macrobrachium gramiimanus, there are no introduced predators in this stream (M. Kido, pers. comm.). The absence of predation pressure on E. sandwicensis likely allows these fish to delay the onset of maturity. Also, high quality food resources may contribute to a high growth rate, allowing fish to reach a large size before initiating the onset of maturity.

Alternatively, fish in Nuuanu Stream may have an earlier onset of maturity and mature therefore at a smaller size. There is a well-established population of non-native smallmouth bass (Micropterus dolomeut) in Nuuanu Stream (Yamamoto and Tagawa

2000). Predation by smallmouth bass and other predators could provide selection

16 pressure for an earlier onset of maturity simply because if fish delay maturity for too

long, they may be eaten before they reproduce (Coates 1992).

Another possible explanation for the difference in size between Nuuanu Stream

fish and Limahuli Stream fish is that poor quality food resources in Nuuanu Stream may also inhibit growth thus resulting in a smaller size at maturity. Thus, it may not be that

fish in Nuuanu Stream are maturing at a younger age, but simply that they are not growing as fast as they would in an environment with higher quality food resources.

These ideas should be explored further. Small sample sizes from the other streams prevent any concrete conclusions from being made at this time. More collections from these streams, as well as age vs. size data for E. sandwicensis and food quality data from

each stream, will yield more useful information, and will better allow us to probe the

effects of environmental quality on the biology of E. sandwicensis.

17 Table 1. Number, ranges of standard lengths (SL), mean standard length, ranges of total wet weights (ww), and mean total wet weight of male and female Eleotris sandwicensis collected from Nuuanu Stream on the island of Oahu, between July 2004 and December 2005.

Number collected SL (mm) Mean SL (mm) ww(g) Meanww(g) Males 82 54-122 85.7±15.88 3.45-38.4 13.9±7.83 Females 51 54-111 79±13.81 3.03-27.64 10.95±6.12

18 Table 2. Histological descriptions of oocyte stages in Eleotris sandwicensis following Wallace and Selman (1981) and Cole (2002). Stage Diameter Characteristics

(rrun)

I (chromatin 0.033- Small; intensely hematoxylin-staining cytoplasm. Few nucleolar) 0.050 nucleoli close to the nucleus periphery.

II 0.030- Intensely hematoxylin-staining cytoplasm. Manydarldy

(perinucleolar) 0.075 staining nucleoli close to the nuclear periphery. ill (cortical 0.058- Pale-staining cytoplasm. Oil droplets begin to appear as alveolar) 0.158 clear vacuoles surrounding the nucleus, and sometimes

form a clear border entirely surrounding nucleus.

Numerous darldy-staining nucleoli in the space between

the cytoplasm and the nucleus.

IV 0.152- Histologically prepared oocytes: majority of cytoplasm is

(vitellogenic) 0.434 comprised of vitelline granules, appeared as pink spheres

in the cytoplasm. Whole oocytes: larger than stage I II

and ill. Look translucent; show spherical shaped globules

in the cytoplasm.

V (hydrated) 0.280- Flat and pink with numerous clear spheres along the

0.500 periphery.

19 Table 3. Number of specimens, range of standard length (SL), mean standard length, range of total wet weight (ww), and mean wet weight of male E. sandwicensis collected from Nuuanu Stream, Wailoa River, Hanamaulu Stream, Kahana Stream, Hakalau Stream, and Limahuli Stream.

Condition Stream Island N SL(mm) meanSL(mm) ww(g) meanww(g) Nuuanu Oahu 82 54-122 85.7±15.88 3.45-38.4 13.9±7.83

Degraded Wailoa Hawaii 20 75-155 100.9±20.48 8.5-38.37 23.08±14.69

Hanamaulu Kauai 9 72-148 108.1±26.98 9.49-66.77 29.44±19.87

Kahana Oahu 8 69-130 89.5±23.65 5.78-46.47 17.59±14.97

Pristine Hakalau Hawaii 11 67-138 104.9±20.36 5.12-41.83 23.83±12.42

Limahuli Kauai 23 94-175 133±18.9 17.66-127.5 52.1±27.S8

20 Table 4. Number of specimens, range of standard length (SL), mean standard length, range oftotal wet weight (ww), and mean wet weight offemale E. sandwicensis collected from Nuuanu Stream, Wailoa River, Hanamaulu Stream, Kahana Stream, Hakalau Stream, and Limahuli Stream.

Condition Stream Island N SL (rom) mean SL (rom) ww(g) meanww(g) Nuuanu Oahu 51 54-111 79±13.81 3.03-27.64 10.95±6.12

Degraded Wailoa Hawaii 22 68-120 97.7±15.05 6.17-37.91 21.16±9.78

Hanamaulu Kauai 16 73-135 98.3±16.19 7.34-45.11 20.06±10.43

Kahana Oahu 10 68-134 92.2±19.54 4.88-64.51 18.9±17.52

Pristine Hakalau Hawaii 12 84-143 114.3±17.59 9.74-41.91 26.81±9.95

Limahuli Kauai 28 92-158 119±17.86 19.05-77.15 36.29±15.70

21 A.) Stage I oocytes (chromat in nucleolar) B.) Stage II oocytes (perinucleolar) C.) Stage III oocytes (cortical alveolar) D.) Stage IV oocytes (vitellogenic) E.) Stage V oocytes (hydrated)

Figure I. Eleolris sandwicensis oocytes sectioned at 711m and stained wi th hematoxylin and eosin (40x).

22 A.) immature stage testis B.) intermediate stage testis showing presence of a few free sperm C.) mature stage testes showing free sperm packing the testis lobules

Figure 2. Eleolris sandwicensis testes sectioned at 7~m and stained with hematoxylin and eosin.

23 oocytes ~~;;t;-;~~ nucleolar) B.) Stage n oocytes (perinucleolar) C.) Stage IU oocytes (cortical alveolar) D.) Stage IV oocytes (vitellogenic) E.) Stage V oocytes (hydrated)

Figure 1. Eleotris salldwicellsis oocytes sectioned at 7fl.m and stained with hematoxylin and eosin (40x).

22 stage stage testis showing presence of a few free sperm C.) mature stage testes showing free sperm packing the testis lobules

and eosin.

23 Size at maturity Nuuanu female E. sandwlcensls 9 8 • 7 • 6 • ... 5 • • • 19 • 4 • • • • •• 3 • • • • • • • • • • • • 2 • • • • .- • • 1 • . " • • • 0.67 .. - ..• • 0 50 60 70 80 90 100 110 Sl (nm)

Figure 3. Size at maturity of female E. sGlldwicellsis. Arrow shows the smallest femal e collected with mature gonads (which was also the smallest female collected overall) Reference line shows the critical aSI for maturity as d.iscussed in the results section.

24 Size at mabJrity Nuuanu male E. sandwlcensis 2.5 •

2.0 • • • • • • 1.5 • • • m :. • •• • • • • • • 1.0 • • • • • • • ••• • ••• • .'•• • •• 0.5 I • ••• I • • • •• • • • •• • • • • 0.204 • •• • • 0.0 • • .' • 50 60 70 80 90 100 HO 120 130 Sl(nm)

Figure 4. Size at maturity of male E. sandwicensis. Arrow shows the smallest male collected with mature gonads (which was also the smallest male collected overall). Reference line shows the critical GSI for maturity as cliscussed in the results section.

25 Spawning season of females 9 8 • 7 • 6 • .. 5 • • t9 • • 4 • • 3 • • • • • • • • • • 2 • • • • • 1 • • I • • • • 0.67 0 • • • -~ -<;t -~ ,~ -~ <,~ -\)<, -\)<, -\)<, -\)<, -\)<, -\)<, -\)<, -\)<, -\)<, .x..\)<' ;;\)<, -\)<, ~#~~~~~~ ~~#~ ~ ~~ ~~~ date of collection

Figure 5. Spawning season of fe male E. SGlldwicellsis. Reference line shows criti cal GS I fo r maturi ty.

26 Spawning season of males 2.5 •

2.0 • • • • 1.5 • • ... • • • I!l • • • • • • • 1.0 • • • • • • • • • • • • • • • • • • • • 0.5 • • I • • • • • • • • • • ! . • • 0.204 0.0 • • • #~~~~~~~~~~~~~~~~~ "V ,?:;;,'\"","l (fr+0 <:!''' "",,,, "'V>~,,} '?-~ ~'" "",,,, "V ,?-",o,,,,,,,"lo 1"" <:!' date of colection

Figure 6. Spawning season of male E. sandwicensis. Reference line shows critical OS! for maturity.

27 Standard Length (SL)

SL (mm)

..... ,

Figure 7. Comparison of standard length among E. sandwicensis collected from 6 streams on 3 islands.

28 Wet Weight -

Wet Weight (g)

I-o :raded. ine l

Condition

Males

Figure 8. Comparison of wet weight between E. sandwicensis collected from 6 streams on 3 islands.

29 Appendix 1. Size frequency histograms of the diameters of 100 vite\logenic oocytes measured from female Eleotris sandwicensis collected from Nuuanu Stream.

N131 - Female 1 " Min. Diam. 0.202mm I- 5 r- Mean Diam. 0.351 mm Max Diam 0.434mm 0 r- - -h GSI8.ll 5 l 0 r-fln O.UI2 0.1818 a.24M 0.3030 0.3636 0.4242 oocyt. dIIIlMt... (1'111'1'1)

N128 5 - Female 2 0 - Min. Diam. 0.192mm 5 - Mean Diam. 0.342mm r- Max Diam 0.424mm 0 f- r- GSI7.04 5

0 r--ri h--r- 0.1211 0.1616 0.2020 0.242" 0.2828 0..3232 O.J6J6 0.4040 oocyt. d • ....t.~ (_)

NI09 - - 5 Female 3 0 Min. Diam. 0.242mm - 5 - Mean Diam. 0.335mm Max Diam 0.404mm 0 GSI5.72 5

0 1 11- ---.-. 0.24 0.27 0.30 0.33 0.36 0.39 oocyte. cIIIo __• (_>

30 .s, » r- " r- Female 4 ,. Min. Diam. 0.202mm ., - Mean Diam. 0.297mm r-: Max Diam 0.404mm ", GSI5.12

0 I rn 0.12 12 0. 161 6 0.2020 1l.1111 0.2828 IU2J2 O.J6l6 0.40411 -rt. dIIo...t.t (.-,)

.23 , - Female 5 • ,- Min. Diam. O.l72mm , - Mean Diam. 0.270mm Max Diam 0.354mm J',0 , GSI4.64 Il-, 0 m- 0.1112 0.1616 0.2020 0.2121 0.2128 0.3232 O.363fi 0. ..0.0 ooqt...... ur(MIt)

N121 - " Female 6 '" r- Min. Diam. O.I92mm " Mean Diam. 0.297mm , ~ r-r- r-: Max Diam 0.364mm , GSI4.36 -rf h Q.U12 0.1616 0.2020 0.2424 o.zan 0.3231 O.l6J6 0.4040 oocyUo ---•• (rmIJ

Nl04 " -r- , " ,- Female 7 • r-r- Min. Diam. 0.152mm - Mean Diam. 0.274mm -r- Max Diam 0.424mm • - , GSI4.099

0 r O.11H 0. 161 6 0.2020 O.HH 0.2828 O.3m O.l63fi m0.40'10 oocytl dllonwt., (!mil

31 NI0S r- • r--- Female 8

> Min. Diam. 0.152mm - --, Mean Diam. 0.322mm J,• f- Max Diam 0.404mm - aSI3.83 s - .-l I I h h •0.1212 0. 1616 0.2020 0.2424 0.1428 0.3232 0.3636 0.4040 oocytl cM. ...tlf (_I .s. " - r- ,. Female 9 Min. Diam. 0.253mm Mean Diam. 0.319mm " - , r-_ Max Diam 0.384mm , ;- - as! 3.61 n---, h 0.1212 0.1818 0.2424 0.3030 0.l6J6 ooq't...... r( ..... ) .t. " - " Female 10 " - Min. Diam. O.I92mm " f- Mean Diam. 0.270mm J" Max Diam 0.354mm .. as! 3.33 , f- r-rf ~ 0. 1212 0.1616 0.2020 0.2424 G.U2a 0.3232 0.3636 0 .~040 oocyte ...... r (MOl)

N12l

S - Female II , Min. Diam. 0.182mm ; • - S Mean Diam. 0.293mrn } ' - Max Diam 0.384mm • r-_ f- aSI3.25 s -rf h •0.1212 0.1616 0.2020 0.2<424 0.2828 0.3232 0.3636 0.40010 oocyt. 6met•• (inti)

32 ... " , ,.. - Female 12 ,. Min. Diam. O.I92mm - Mean Diam. 0.268mm ~ J Max Diam 0.333mm , ;- aSI3.16 5

0 ...... r 0.1212 0.1616 O.102a 0.2424 0.2828 rn0.3132 0.3636 O.~ oocyte "1IIIt.r (MIl)

H110 " » , Female 13 ".,. Min . Diam. 0.182mm -- Mean Diam. 0.274nun J" ,. C- Max Diam 0.333mm as! 2.71 > n-r 0.1211 0.1616 O,lOlO 1U414 O.ltll o.J212o O.l6l6 0. ..040 --rte cIIMIter ( ~

N4' " Female 14 Min. Diam. 0.182mm Mean Diam. 0.303mm Max Diam 0.364mm as! 2.697

~~."~"~OC. "~"~O~±=~.±M2~' ~'2±"~'~.~±~~~'~~~O~.~~J oocyte~ ... (- l

N6

" ~- " 25 Female 15 ,. Min. Diam. O.l92mm , Mean Diam. 0.272mm " Max Diam 0.3 23mm " - 5 as! 2.68 J h 0. 1212 0.1616 0.2020 0.2424 0.2'la o.J1l2 0.3636 0,4040 o«yte cllllI'IIt., (IIWI,)

33 N116 )0 r- 25 Female 16

20 Min. Diam. 0.182mm

-~ c- Mean Diam. 0.273mm I ~ Max Diam 0.343mm o OSI2.56 5 Iff rh 0. 1212 0.1616 0.2020 0.2424 0.282:8 0.3232 0.3636 0.'t(MQ ooo:yU dlalllft.r (_)

N1ll JO r- 25 Female 17 20 Min. Diam. 0.182mm " r- - Mean Diam. 0.290mm , Max Diam 0.354mm -_r- - OS! 2.52 5

0 r-r- 0. 1212 0.1616 0.2020 0.2424 0.2828 0.3232 0.3636 0.4040 oocyt. dalllft.r (_ )

34 Appendix 2. Standard length, wet weight, Gonadosomatic index (OSI). and month of collection of female Eleotrls sandwicensis collected from Nuuanu stream between July 2004 and December 2005. SL (rom) ww(g) GSI Month 86 17.23 2.15395 July 78 8.3657 2.68442 July 109 23.689 3.25559 Sept 96 16.6908 3.28849 Sept 69 6.2085 3.09869 Oct 75 15.7594 3.24555 Oct 72 11.7623 4.64307 dec 69 5.1325 1.32467 dec 58 3.0319 1.65292 Jan 75 6.6211 5.18699 Jan 94 15.8934 1.09534 Jan 64 3.9161 1.57442 Jan 85 18.1362 3.86988 Jan 91 14.448 1.79953 Jan 67 6.3287 1.7885 Feb 75 6.812 2.69708 Feb 71 6.3997 1.1027 Mar 111 14.5875 0.8176 Mar 74 8.3131 0.6904 Mar 66 5.7644 0.53368 Apr 82 9.4126 1.15637 Apr 72 7.813 0.38804 May 65 5.6161 0.56945 May 74 7.2896 0.92066 May 64 4.958 1.05787 May 100 23.7082 3.15627 Jun 85 13.5854 5.11517 Jun 109 27.6425 5.11775 Jun 72 8.9303 2.6141 July 91 16.3937 3.61332 July 108 26.8417 2.14786 July 85 12.3465 2.12497 July 77 8.7496 1.42465 Aug 63 4.6146 0.53376 Aug 76 8.4355 1.27988 Aug 92 16.5114 3.33252 Aug 83 12.3306 2.01877 Aug 89 10.8173 4.09858 Sept 82 11.0502 3.82696 Sept 87 11.8409 0.53917 Sept

35 Appendix 2 continued

SL (rom) ww(g) GSI Month 75 9.1293 5.71703 Sept 75 7.95 2.70522 Sept 97 18.145 2.52223 Oct 65 5.0489 2.56155 Oct 54 3.2638 3.01749 Oct 76 8.1577 4.36379 Oct 89 17.4894 7.03755 Nov 75 9.2937 1.82645 Nov 70 7.2272 8.10584 Nov 61 4.0722 0.45637 Dec 64 4.6707 0.67032 Dec

36 Appendix 3. Standard length (SL), wet weight (ww), Gonadosomatic index (GSI), and month of collection of male Eleotris sandwicensis coIlected from Nuuanu stream between July 2004 and December 2005.

SL (rom) ww(g) GSI Month 95 18.49 0.65324 Jul 87 11.995 0.55414 Jul 89 15.42 1.19372 Jul 104 21.418 0.81195 Jul 72 6.6 1.03794 Sep 102 18.71 0.7501 Sep 106 25.143 1.21614 Sep 112 26.285 1.16464 Sep 110 20.353 0.27838 Oct 75 7.619 1.63001 Oct 84 11.227 2.46331 Oct 72 6.893 0.35522 Oct 103 16.753 1.664 Dec 92 7.851 0.89188 Dec 62 4.503 0.10226 Dec 115 27.472 1.23073 Dec 61 4.279 0.49558 Jan 63 3.996 0.88118 Jan 98 16.707 0.83591 Jan 82 11.239 0.80636 Jan 54 3.552 0.36447 Jan 57 3.445 0.15411 Feb 87 11.374 1.00257 Feb 69 6.165 0.29608 Feb 94 6.753 1.42379 Feb 87 8.503 0.48451 Feb 107 21.007 0.65306 Mar 120 38.37 1.21634 Mar 95 17.454 0 Mar 86 10.83 0.3735 Mar 104 29.344 1.11472 Mar 104 23.935 0.47478 Mar 80 10.362 0.52584 Apr 66 5.598 0.03217 Apr 79 12.633 0.60444 Apr 70 6.051 0.40818 Apr 82 10.646 0.55727 Apr 80 10.095 0.22935 Apr 73 7.024 0.15401 Apr 85 12.402 0.1777 May

37 AI.ppen dix3 continu. ed SL (mm) ww(g) GSI Month 75 10.518 0.20388 May 105 25.592 0.9045 May 95 20.301 0.70839 Jun 85 13.239 0.36617 Jun 86 14.609 0.83449 Jun 122 36.993 1.1874 Jun 90 16.87 0.68576 Jun 110 30.694 1.11744 Jun 92 17.282 1.73299 JuI 80 12.293 1.52295 JuI 85 12.107 0.87233 JuI 83 12.399 1.24192 JuI 85 11.918 0.97175 JuI 100 22.624 1.28531 Aug 83 11.24 1.30783 Aug 64 5.098 0.77691 Aug 82 11.538 0.99618 Aug 68 6.632 1.43624 Sep 87 12.326 0.68862 Sep 92 16.394 0.49593 Sep 62 5.083 0.28215 Sep 90 14.882 1.08409 Sep 80 10.716 0.41796 Sep 85 12.963 1.99781 Oct 82 11.396 1.25457 Oct 103 22.017 1.22385 Oct 60 3.915 0.37688 Oct 61 4.488 1.85425 Oct 59 4.051 0.93938 Oct 80 11.291 1.20825 Nov 67 6.353 1.04814 Nov 97 22.336 1.23873 Nov 102 21.289 0.86896 Nov 67 6.239 0.56089 Nov 80 12.216 1.3389 Nov 91 16.833 2.06274 Nov 64 4.96 0.27497 Dec 90 15.077 0.39555 Dec 80 12.116 0.42936 Dec 89 14.076 0.28069 Dec 103 24.355 0.93789 Dec 107 24.276 0.46391 Dec

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42