The Effects of Introduced Fish Species on the Endemic Shrimp of Anchialine Ponds

THE EFFECTS OF INTRODUCED FISH SPECIES ON THE ENDEMIC SHRIMP OF ANCHIALINE PONDS

Meredith F. Acly Senior Thesis Department of Marine Science University of Hawaii at Hilo

May 6,2003

Advisors: Dr. Karla McDermid and Dr. Cedric Muir Abstract

Anchialine ponds, or tidally influenced coastal ponds, are common along the coasts of

Hawai 'i Island. yet rare worldwide. The endemic species Halocaridina ruhra (Red Pond

Shrimp) when in high abundance, can create red carpets along the bottom of anchialine ponds.

Unfortunately, many ponds contain introduced species of fish which reduce or displace native

shrimp populations. Of the estimated 650 anchialine ponds on Hawai'i Island, 300 were

surveyed in this study. Forty-three percent of the ponds no longer contain evident shrimp

populations. Twelve ponds were visited monthly from August 2002 to April 2003 to measure

shrimp density, and physical parameters in the presence and absence of Poecilia reticulata

(Trinidadian Guppy), an introduced fish. Shrimp densities in the presence of fish were

significantly lower. In the presence of fish, various unusual behaviors were observed such

asshrimp present only in cracks or crevices at pond edges. The rate and frequency of

subterranean shrimp migration between ponds due to introduced fish or natural behaviors is

unknown. The study included preliminary work to extract, amplify. and sequence DNA from

individuals living in different ponds. By comparing the frequency of alleles in different ponds

one can assess the degree to which gene flow (migration) occurs between these ponds.

Introduction

The island of Hawai'i, like all Hawaiian Islands, faces extinction of native species and damaged ecosystems due to habitat loss and the introduction of exotic species. Exotic species introduced into Hawaiian ecosystems typically lack natural predators, resulting in fast-growing populations existing alongside and out-competing native species. These processes have occurred on land and in nearshore habitats, resulting in an extinction of numerous plant and animal

species.

One ecosystem in particular, anchialine ponds, though common around the island of

Hawai'i, is rare worldwide. Characterized as land-locked brackish water pools, this distinctive habitat does not have surface connections to ocean water, but connects directly to the sub-surface water table, which controls daily tidal fluctuations in the ponds (Brock, 1985). Anchialine ponds are most common throughout the newer lava flows of the Kona coast on Hawai'i and Maui

Islands, as well as a bomb crater on Kaho'olawe, a lake on Moloka'i, and in limestone formations on O'ahu (Bailey-Brock and Brock, 1993).

Over the last twenty years, the loss of suitable anchialine pond habitat has totaled more than 95%, making the habitat one of the rarest in the country (Brock, personal communication,

2001). The most common source of degradation has been the introduction of alien fish species, mainly Oreochromis mossnmbicus (Tilapia), and various types of topminnows including

Gambusia affinis (Mosquito Fish), Poecilia reticulata (Trinidadian Guppy), and Poecilia mexicana (Shortfin Molly). Intentional introduction of an exotic fish may be for its later harvest as fish bait, insect control, or food; accidental introductions can occur when one pond in a system is intentionally stocked and the introduced species colonizes other ponds (Brock, 1985). Once topminnows have been introduced into one anchialine pond, fish eventually migrate to ponds in close proximity, traveling through porous basalt walls separating pond clusters (Chai, personal communication, 2002).

Other sources of degradation include human use and impact. Construction of resorts and small industrial developments along popular coastal stretches has already caused an unknown quantity of ponds to be filled in. At least 130 ponds were destroyed in 1985, during the construction of the Waikoloa Resort Village (Brock et al., 1987). Resorts have also increased human accessibility to some ponds. resulting in decreased pond water quality or the further destruction of ponds. Ponds have become bathtubs or trash receptacles, and most are impacted by fertilizers and chemicals washed down from golf courses, malls, and homes. Over time pond awareness has increased, and now all coastal development near anchialine pools must now include detailed pool inventories and management plans (Chai, 1993). Numerous ponds have been surveyed on land and using aerial photography, and between 600 and 650 were been counted (Brock, 1985). The estimate is conservative, as there may be hundreds of ponds on the island undetectable from the air, or completely unknown to scientists. Exact numbers of ,. anchialine ponds and their health status is crucial information in the preservation of this native habitat.

Among many unique flora and fauna, Hawai'i's anchialine ponds are home to endemic shrimp species, the two most common being Halocaridina rubra (Family Atyidae) or Opae 'ula and Metabetaeus lohena (Family Alpheidae). The shrimp are hypogeal, meaning that they utilize both the pond habitat as well as subterranean rock interstices linking ponds to the underIying water table. Many of the shrimp display a tidally linked migration, emerging and returning to rock interstices with the incoming and ebbing tide (Brock, 1985). Shrimp have been recorded with higher abundances during night hours versus daylight hours, indicating a definite preference for nocturnal activity (Chai, 1993). H. rubra is characterized as a small, red

L herbivorous shrimp commonly seen grazing along the algal substrate of the ponds. The larger M.

Iohena reaches lengths of approximately 16 mm, about twice the size of H. rubra, and is most often observed hunting H. rubra (Banner and Banner, 1960). Typically M. Iohena and H. rubra exist in a 1 :100 ratio. * No native fish are known to successfully reproduce in anchialine ponds (Brock, 1992) but in recent years, it is suspected that the total number of anchialine ponds containing reproducing introduced fish species has increased. The actual effect introduced fish have on anchialine shrimp is only hypothesized, but many believe fish prey upon native shrimp. Studies have indicated lower or absent populations of shrimp exist when introduced fish are present (Brock,

1985; Chai, 1993). In order to return native ponds back to anchialine species, information on the total numbers of anchialine ponds containing alien fish must be determined, and data on how these fish affect shrimp distribution and abundance is needed. Few ponds have been observed where shrimp and introduced fish co-exist, but Brock (1 985) calculated shrimp to inhabit 18% of the ponds containing introduced fish in the one study. In the absence of herbivorous H. rubra, ponds are overgrown by algae which has the potential to fill in ponds (Brock, 1992).

Ponds over run by introduced fish can be chemically treated to destroy introduced fish and restore the habitat. In one treatment of rotenone, shrimp returned to the fish-free ponds

(pers. obs., 2002), thus exhibiting their hypogeal nature. It is unknown to what extent shrimp move freely between undisturbed ponds and locations. Morphological differences in cheliped shape, spination, length of pereopod joints, and rostrum length (Bailey-Brock and Brock, 1993) have been noted between samples of H. rubra raising the question of possible genetic differences between anchialine pond populations.

To date, published work on anchialine ponds and shrimp have been general surveys of the physical and chemical aspects of ponds (Aquatic Resources Management and Consulting,

1994, 1995; Brock, 1985; Brock, 1985; Brock et al., 1987), and the description and classification of the known endemic shrimp species (Bailey-Brock and Brock, 1993; Banner and Banner, 1960;

Couret and Wong, 1978; Holthius, 1973; Kensley and Williams, 1986) Methods

Thirteen anchialine ponds located along the Kona coast of the island of Hawai'i (Figure

I) were visited from July, 2002 to April, 2003. Temperature, salinity, dissolved oxygen, and shrimp density were recorded at each visit. Three ponds at each of the sites, Kaloko-Honokohau

National Historical Park (KAHO), Four Seasons Resort, Waikoloa Resort Village (Waikoloa

Anchialine Pond Preservation Area (WAPPA)), and four ponds at Makalawena Beach were included in this study.

Physical parameters were measured using a YSI Model 85, which tests temperature, salinity, and dissolved oxygen. Measurements were taken at the bottom of the pond, or if the pond is deep, at five meters depth which is the length of the YSI cable. Shrimp density was taken using a 10 X 10 cm quadrat placed randomly around the circumference of each pond. The edges of the pond were tested to prevent destruction of fragile algae often growing throughout -. _. the center of the ponds. At each pond, five density counts were taken: the number of shrimp inside each quadrat during the span of one minute. The five counts were averaged on each sampled day and used in comparisons between ponds. Shrimp density at Makalawena Beach was estimated by counting visible shrimp around the entire circumference of the pond. The circumerference is defined in this study as the perimeter into one half meter of the pond. This - --- area of each pond at Makalawena Beach was measured, and shrimp density was calculated to shrimp/m2. Shrimp densities at all other locations were extrapolated from 10 x 10 cm quadrat counts to shrimp/m2 densities.

Pond across the island were located using a previous aerial survey (Biological Database, -- 1987). Latitude, longitude, and estimated position error of each pond were recorded using a

Gamin eTrex Legend handheld GPS. Temperature, salinity, and dissolved oxygen were taken - using a YSI, and species present were noted. All surveying was performed during daylight

hours.

Tidal delay for anchialine ponds was approximated using data from one pond in Hilo. A

PVC pipe with marked intervals was placed standing up in the middle of the pond at the time of

high tide. The still rising water was observed until it began to fall, and the difference between

coastal high tide and the pond's high tide (tidal delay) was used in later analysis.

Shrimp migration between ponds on the island of Hawai'i was examined by sequencing

the mitochondria1 cytochrome oxidase subunit one (C01) gene of individual shrimp. Due to the

high degree of mutations of C01, ten or more individual shrimp from each pond were targeted to

sequence as to account for natural mutation rates in a population. Shrimp were caught from each

selected pond and frozen individually in pond water. DNA was extracted from each individual

thawed shrimp following the animal tissue protocol of the Qiagen DNEasy Tissue kit (Qiagen,

2002). Extracted DNA was used in a polymerase chain reaction to amplify C01. The following

amounts of reagents were used for the PCR for a total reaction of 25 pL: 2.5 pL PCR buffer, 2.5

pl MgCI2, 2.5 p1 dNTP, 1.5 pL primer 1490, 1.5 pL primer 2 198, 0.5 pL BSA, 5.0 pL Q

solution, 3.0 pL DNA template, 5.75 pL H20, 0.25 pL DNA polymerase.

The reaction was performed using a MJ Research, Inc. PTC-100 Programmable Thermal

Controller using the following program: Step 1, 94 "C for 3.00 minute; step 2, 94 "C for 1 :00

minute; step 3'45 "C for 1.00 minute; step 4, 72 "C for 1:00 minute; step 5, repeat steps 2-4 for

40 cycles; step 6, 72 "C for 7:00 minute. The PCR product was run on an agarose and 0.5~TBE

gel at 40 volts for two hours. Individual C01 bands were excised from the gel.

Excised DNA bands were purified following the protocol of the Qiagen QIAquick kit for gel extractions using a microcentrifuge (Qiagen, 2002). Purified DNA was used in a reaction - using the following reagents for a total of 18 pL: 2.0 pL reaction buffer, 1.5 pL dNTP, 1.25 pL

primer 1490,4.0 pL DNA, 0.5 pL l00X BSA, 2.0 pL DNA polymerase, 6.75 pL H20. 4.25 pL

of the solution were added to 4 pL of dGTP, dATP, dTTP, and dCTP. The reaction was

completed using a thermocycler programmed to: Step 1,94"C for 3:00 minute; step 2, 94°C for

I 1 :00 minute; step 3,45 "C for 1 :00 minute; Step 4, 72 "C for 1:00 minute; step 5, repeat steps 2-4 1 for 40 cycles; step 6, 72 "C for 7:00 minute. Upon completion, 4.0 pL of stop solution was added to the reaction. The reaction was then denatured using the thermocycler programed to 94

"C for 3:00 minute. The sequencing reaction was immediately loaded onto a gel composed of

40 mL 5.5% KB''"' modified acrylamide. 270 pL APS, 27 pL TEMED, using a LiCOR DNA

Analyzer Gene Readir 4200 sequencer set on 41 cm and 700 channel electrophoresis condition.

Results

The four ponds at Makalawena Reach all contained H. rubra and M. lohena, as well as

the introduced fish, Poecilia reticulata (guppy). Ponds were assigned labels; Pond One, Pond

Two, Pond Three, and Pond Four. The sea grass Ruppia maritima grows in the soft sediment of

I the center of the Pond One, while the edges of the pond are rocky. H. rubra were only observed

at pond edges, while P. reticulata were observed in all parts of the pond. Pond One lies 248 feet

from the cluster of ponds Two, Three, and Four. Pond Two is surrounded by a'a lava, at the

edge of a cluster of kiawe trees. Sediments line the center of the pond, and a'a at the pond edges.

H. rubra were only observed on top of and inside of these rocks, while P. reticulata swam in all

sections of the pond.

The Kiawe trees marked the beginning of a pahoehoe lava flow. H. rubra were observed

I along all pond surfaces. M. lohena were always observed amongst H. rubra, sometimes in - contains few rocks, but rather soft sediments. Both rocks and sediments were not covered by macro-algae. Only a small patch of R. maritima lies at one end of the pond. Both H. rubra, M. lohena, and P. reticulata were observed in all portions of the pond, while M. lohena was often present in equal abundance as H. rubra. For all ponds at Makalawena, no attempts at estimating fish density or ratios between H. rubra and M. Iohena were made.

Temperature and salinity in the four ponds at Makalawena did not have corresponding highs and lows. Ponds One, Two and Three, Four coupled in both the salinity (Figure 2) and temperature (Figure 3) to follow very similar trends. Dissolved oxygen showed similarities between ponds, mostly between Ponds One, Two, and Three (Figure 4). Shrimp densities for the four ponds all followed different trends throughout the study period. Shrimp density in Pond

One had highs and lows corresponding with those of salinity, while density of shrimp in Pond

Two was dictated by tide height (Figures 5,6). Shrimp density in both Ponds Three and Four was eratic and does not obviously follow any trends (Figures 7,8).

The three ponds at Waikoloa Anchialine Pond Preserve Area all contained H. rubra, M. lohena, and the epigeal shrimp Paleomon debilis. Ponds numbers were kept consistent with a

1992 study (Brock, 1992) of Ponds 48,49, and 50. All three ponds are surrounded by a'a lava.

H. rubra and P. debilis were dispersed across entire pond bottoms, and on two occasions P. debilis were observed eating H. rubra. M. lohena were only observed during twilight hours.

Ponds were in close proximity, with distances of five to ten feet separating each.

Salinity of the three ponds at WAPPA were similar on each sampling day, not varying by more than 2.5 ppt (Figure 9). Temperature was also similar between the three ponds (Figure 10).

Dissolved oxygen also followed the same increase and decrease trend between ponds (Figure

11). For all three ponds, comparisons of shrimp density, tidal height, and salinity show different - contains few rocks, but rather soft sediments. Both rocks and sediments were not covered by macro-algae. Only a small patch of R. maritima lies at one end of the pond. Both H. rubra, M. lohena, and P. rericulata were observed in all portions of the pond, while M. Iohena was often present in equal abundance as H. rubra. For all ponds at Makalawena, no attempts at estimating fish density or ratios between H. rubra and M. lohena were made.

One had highs and lows corresponding with those of salinity, while density of shrimp in Pond

Two was dictated by tide height (Figures 5,6). Shrimp density in both Ponds Three and Four was eratic and does not obviously follow any trends (Figures 7,8).

The three ponds at Waikoloa Anchialine Pond Preserve Area all contained H. rubra, M. lohena, and the epigeal shrimp Paleomon debilis. Ponds numbers were kept consistent with a

1992 study (Brock, 1992) of Ponds 48,49, and 50. All three ponds are surrounded by a7alava.

H. rubra and P. debilis were dispersed across entire pond bottoms, and on two occasions P. debilis were observed eating H. rubra. M. lohena were only observed during twilight hours.

Ponds were in close proximity, with distances of five to ten feet separating each.

Salinity of the three ponds at WAPPA were similar on each sampling day, not varying by more than 2.5 ppt (Figure 9). Temperature was also similar between the three ponds (Figure 10).

Dissolved oxygen also followed the same increase and decrease trend between ponds (Figure

11). For all three ponds, comparisons of shrimp density, tidal height, and salinity show different high North water. Temperature of the three ponds was very variable on any one given day

(Figure 16). Shrimp density in the North Pond did not follow tide heights; rather it followed the

salinity of the pond (Figure 17). Density at the Middle Pond. like WAPPA's Pond 49, followed

and disobeyed all trends (Figure 18). Days of low shrimp density were at a high 1.7 foot tide as

well as a low 0.0 foot tide. The highest average shrimp density at the Middle Pond was 5 180

shrirnp/m2 which occurred on a 2.6 foot tide. The next highest density day was 5000 shrimp/m2

which corresponded with a 0.0 foot tide.

The ponds at Kaloko-Honokahau National Historical Park were labeled Ponds 83, 84, and

85, consistent with previous labeling by the park. The ponds created a triangle on a pahoehoe

field, with 33.3 feet separating Ponds 83 and 85, 70.9 feet separating Pond 83 and 84, and 53.5

feet separating Ponds 84 and 85. H. rubra were dispersed across pond bottoms at all three

ponds, and M. lohena were observed on two occasions in Pond 85. Ponds 83 and 85 are rock and

thin sediment bottoms, and Ulva grows across the surface of the pond. Pond 84 does not contain

evident macro algae. rather a population of California grass lies at the pond perimeter.

Salinity and temperature of the three KAHO ponds was very consistent between ponds

during the study time (Figures 20,2 1). Dissolved oxygen, as with other surveyed ponds, was

highly variable showing no trends (Figure 22). The salinity of all three ponds followed the highs

and lows of tidal heights, except on March 16,2003. This date marked the lowest tide heights

(-0.1 to 0.05) and the highest recorded salinity for all three ponds.

Data from 300 ponds across Hawai'i Island was collected from June, 2002 to April, 2003.

Sampled ponds are located on all coastlines of the island (Figure 22) except for the older northeast coastline which no longer has coastal lava beds essential for anchialine ponds. Jessica

Schwatz of Kaloko-Honokohau National Historical Park provided information on 57 ponds - residing in the park. The author collected the remaining data. It was found that shrimp inhabited

ponds with a range in salinity from I .7 to 24.7 ppt, temperature from 1 8.0 to 36 OC, and dissolved

oxygen from 3.13 to 11.81 mg/L. Fish inhabiting ponds were found in salinities from 2.8 to 12.2

ppt, temperature from 23.2 to 3 1.8 OC, and dissolved oxygen from 3.04 to 8.45 mg/L. Many

species of fish were observed, especially in residential areas containing ponds. In these backyard

ponds, fish species ranged from Tilapia to Koi, to Manini, to Ulua. Residents frequently bring in

new fish from the reef to stock their ponds, and very few protected their ponds from fish. Only

5.3% of ponds containing fish were found to contain evident shrimp populations. Across the

island, H. rubru are no longer found in 43% of the surveyed ponds that either contain fish

species, or no fish or shrimp species.

The most common fish observed during the study are freshwater topminnows or guppies,

mostly P. reticulafa. Ponds close behind beach berms were also observed to contain coastal fish

such as Aholehole and Manini which were most likely thrown into the ponds during storms and

high surf. Ponds containing these relocated marine species did not show the presence of juvenile

fish. Ponds containing freshwater guppies contained all sizes of fish, from juveniles to adults

and fish were abundant across the pond.

\eucemly six ponds across the island have successful DNA extractions performed from

their shrimp. Pond 83 from KAHO has generated five sequences of between 61 0 and 650

nucleotides. Of these sequences eight nucleotide differences between individuals has been

noted. Using a GenBank database (Altschul et. A].. 1997), one H. rubra C01 sequence of 641

/ nucleotides most significantly aligned with C'herax fenuimanus. Cherux is a freshwater crayfish

used in aquaculture. The remaining five ponds can be sequenced once the polymerase chain reaction has been altered to account for individual shrimp's DNA template concentrations. Discussion

Physical parameters of anchialine ponds are highly variable between ponds across the island,

as well as for individual ponds at different tidal heights. The 13 ponds visited since July, 2002

all display different ranges in temperature, salinity, and dissolved oxygen. Tide height

dominates physical parameters for ponds. while tide height, physical parameters, and time of day

all seem to influence shrimp density. The Middle Pond at the Four Seasons resort had its second

highest shrimp density count despite a 0.25 tide. The pond was sampled at 4:45 PM, shwmg

that more shrimp are present in the later afternoon and twighlight hours even with less favorable

tide conditions.

From casual observations of anchialine ponds across the Flawai'i Island, one can easily see

that very few are pristine collections of their endemic shrimp inhabitants. More and more ponds

are now dominated by guppies and other introduced fish species rather than Opae 'ula. Many

ponds in residential areas have contained fish for years, and pond owners are not aware of the

unique habitant these ponds provide. The ranges in temperature. salinity, and dissolved oxygen

meet and exceed those of fish meaning that habitats taken over by fish are still suitable for

shrimp inhabitance. These physical factors are not restricting shrimp from ponds containing fish.

The only natural predator of H. rubra are the larger M. lohena. Like most top predators M. lohena are far less abundant and infrequently observed. No Hawaiian fish are native to anchialine ponds, so with the absence of a large predator fish populations, and in pal+iculk P. reticulata, reproduce uncontrollably. If all epigeal, or surface waters of ponds are filled with fish, it is unknown whether shrimp can survive in only the hypogeal. Ponds no longer containing shrimp are close to 50%, so the question of can the shrimp surface permanently underground may be'asked in the near future. - Luckily there are still many inaccessible or difficult to reach ponds across the island

harboring shrimp populations. Surveyed ponds were termed accessible if they could be visited

via a paved or two-wheel drive dirt road, or are within a 30 minute walk of such a place. Thirty-

four percent of the 350 ponds surveyed are classified as secluded and not easily visited. Of these

ponds, shrimp are still present in 76% of these ponds. For easily accessible ponds the numbers

are nearly reversed, with 67% of easily visited ponds void of shrimp. Ponds in remote parts of

the island may still be safe from accidental or intentional fish introductions for now.

Once fish are residents of anchialine ponds, it is suspected that small guppies can travel short

distances through porous lava to neighboring ponds. This may be the case of the South Pond at

Four Seasons Resort, which resides 50 feet away from an estrablished population of P.

mexicana. Other mechanisms such as water birds may also carry fish over short distances. If

these hypotheses are true, then despite periodic rotenone treatments, fish will ultimately return to

the pond. The positive side is that shrimp return to the pond, either emerging from the

intersticial waters or they migrate from neighboring pond once fish are killed.

DNA sequencing of shrimp populations will provide critical knowledge in understanding

natural behaviors of shrimp, as well as behaviors induced due to introductions of fish. Despite

the exact reason for leaving, data from this study shows that once fish have entered a pond

system shrimp densities are significantly lower or absent. Shrimp which are currently in the

process of being sequenced span the four sides of our island. These ponds are not in the viscinity

of other ponds containing fish, therefore they should feel no un-natural pressure to migrate. The

next step would be to sequence shrimp from ponds close together and still without fish pressure.

WAPPA which has hundreds of ponds all lacking fish, would be a perfect study site. Once these ponds are sequenced, one can asses the degree of potential gene flows from ponds at great - distance. as well as in close proximity. This will determine the natural migratory behaviors of shrimp.

These behaviors can then be compared to those exhibited from the introduction of fish.

Makalawena Beach borders a field of ponds containing a mix of fish and shrimp. Close to the popular beach, P. reticulata have been introduced and dominate these ponds. No shrimp are present. As the distance from shore increases, fish are less frequent in ponds and shrimp appear along with the guppies. Still further from shore, ponds no longer contain P. reticulata and H. rubra is abundant. By comparing shrimp sequences to varying distance from shore, and from the fish pressure at Makalawena Beach, migration due to the fish can be assesed. It is possible shrimp do not migrate away from fish. rather move into the intersticial waters beneath the ponds.

Whatever scenario occurs, these studies will help to predict the reactions of shrimp to increasing fish pressure across Hawai'i Island.

Conclusion

This study has proven that reduced or absent endemic Halocaridinu rubra populations from anchialine ponds are a result of introduced fish. Continued introduction of fish into pondsand the possible short range migration of fish into neighboring ponds will oniyincrease the percentage of ponds lacking shrimp. Education needs to be continued to ensure residents of the island of

Hawai'i are aware of the unique anchialine pond systems, and to help in the protection of their endemic inhabitants. DNA analysis will help in understanding the response of shrimp to fish introductions, as well as in future management plans of the anchialine habitat. Literature Cireed

Altschul, S.F, T.L. Madden, A.A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D.J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.

Aquatic Resources Management and Consulting, Waikoloa, Hawaii. 1994. Anchialine pond biological monitoring and management recommendations. Report 3-94. Kailua-Kona.

Aquatic Resources Management and Consulting, Waikoloa, Hawaii. 1995. Anchialine pond biological monitoring and management recommendations. Report 4-94. Kailua-Kona

Bailey-Brock, J.H., and R.E. Brock. 1993. Feeding, reproduction, and sense organs of the Hawaiian Anchialine Shrimp Halocaridina rubra (Atyidae). Pac. Sci. 47(4):338-355.

Banner, A.H. and D.M. Banner. 1960. Contributions to the knowledge of the Alpheid shrimp of the Pacific Ocean Part VII. On Metabetaeus Borradaile, with a new species from Hawaii. Pac. Sci. 14( 1 3):299-303.

Brock, R.E. 1985. An Assessment of the condition and future of the anchialine pond resources of the Hawaiian Islands. Environmental Assessment Company, Honolulu, Hawaii.

Brock, R.E. 1985. Aquatic survey of the anchialine pond system at Lahuipuaa, Kona, Hawaii. Mauna Lani Resorts, Kawaihae, Hawaii.

Brock, R.E., and A.K.H. Kam. 1992. Waikoloa Anchialine Pond Program: Fourth Status Report. HIMBISeagrant, University of Hawaii, 1992.

Brock, R.E., J. Norris, D. Zeimann, and M.T. Lee. 1987. Characteristics of water quality in anchialine ponds of the Kona, Hawaii coast. Pac. Sci. 4l(1-4):200-208.

Chai, D. 1993. A Biophysical Inventory and Assessment of Anchialine Pools Along the Waiakea Coast, Hilo, Hawaii. MS Thesis, Department of Geography, University of Hawaii, Honolulu.

Couret, C.L., and D. Wong. 1978. Larval development of Halocaridina rubra Holthius (Decapoda, Atyidae). Crustaceans. 34(3):30 1-309.

Holthius, L.B. 1973. Caridean shrimps found in land-locked saltwater pools at four Indo-West Pacific localities (Sinai Peninsula, Funafuti Atoll, Maui and Hawaii Islands) with the description of one new genus and four new species. Zool. Verhand. 128: 1-48.

Kensley, B. and D. Williams. 1986. New shrimps (Families Procarididae and Atyidae) from a submerged lava tube on Hawaii. J. Crust. Biol. 6(3):417-437.

Salinity of Ma kalawena Beach Ponds

Date I I -+- Pond One +Pond Two +Pond Three -8- Pond our/

Figure 2: Salinity of ponds at Makalawena Beach

7 7 Temperature of Makalawena Beach Ponds

I Date

Figure 3: Temperature of Ponds at Makalawena Beach Dissolved Oxygen of Makalawena Beach Ponds

i Date I - -- I -&- Pond One tPond Two --+- Pond Three tpond I I I - Figure 4: Dissolved oxygen of ponds at Makalawena Beach

Makalawena Beach Pond One

1 Date I +Tide Height +Salinrty +Shrimp Density

Figure 5: Shrimp Density, Tide Height, and Salinity Pond One at Makalawena Makalawena Beach Pond Two

Date -- / -m- Tide Height +salinity -t Shrimp El .- Figure 6: Shrimp density, tide height, and Salinity of Pond Two at Makalawena

Makalawena Beach Pond Three

Date

.. I +Tide Height +Salinlty -t- Shrimp Densitv 1

I- Figure 7: Shrimp Density, Tide Height, and Salinity of Pond Four at ~akaiwena Makalawena Beach Pond Four

I Date 1 -. -- I 1 +Tide Height +Salintty -6 Shrimp ~ens@] ---- Figure 8: Shrimp Density, tide height, and salinity of Pond Four at Makalawena

Salinity of WAPPA

Date

tPond 48 +Pond 49 --e- Pond 50 1

Figure 9: Salinity of ponds at Waikoloa Anchialine Pond Preserve Area Temperature of WAPPA

Date I --- I tPond 48 tPond 49 +Pond 50 j -- L. - 1 Figure 10: Temperature of ponds at Waikoloa Anchialine Pond Preserve Area -- - . --

7.- -- I I WAPPA Pond 48

i Date I 1 +Tie Height +Salinrty -+Shrimp Density /

I___- I Figure 11: ~issxedoxygen of ponds at Waikoloa Anchialine Pond Preserve Area Dissolved Oxygen of WAPPA Ponds

Date

Pond 48 -D- Pond 48 +pond -.

- - - Figure 12: Shrimp density, tide height, and salinity of Pond 48 at WAPPA i WAPPA Pond 50

i Date / tTide He~ht +Salinity -t-- Shrimp Density Figurel3: Shrimp density, tide height, and salinity of Pond 50 at WAPPA WAPPA Pond 49

Date I / +Tie Height tSalinity --t Shrimp Density i

Figure 14: Shrimp density, tide height, and salinity of Pond 49 at WAPPA I 7 Salinity of Four Seasons Ponds

I I I I i

Date L---e Middle Pond -m- North Pond -t-- South Pond / Figure 15: Salinity of ponds at the Four Seasons Resort I Temperature of Four Seasons Ponds

Date 1

- -. +Middle Pond +North Pond +South pond1

L-- Figure 16: Temperature of ponds at the Four Seasons Resort

Four Seasons Resort North Pond

Date - -m- Tide Height -o- Salinrty +Shrimp Densrty 1 Figure 17: Shrimp density, tide height, and salinity of the North Pond at the Four Seasons Four Seasons Middle Pond

Date 1 +Tide Height +Salinity +Shrimp Density /

Figure 18: Shrimp density, tide height, and salinity of the Middlle Pond at Four Seasons

Salinity of KAHO Ponds 30

hC,a 20 a Y .-3 15 -.-c m m 10

Q~~O~lQ2 lo12~ lQ2 ,219~1Q2 Q2 1291~~ ~~12~1'~ ~~1091~~

Date

I +Pond 83 +Pond 84 +Pond 85 1

Figure 19: Salinity of pond at Kaloko-Honokohau National Historical Park Temperature of KAHO Ponds

Date ( -+- pond 83 +Pond 84 -t~ond 85 /

Figure 20: Temperature of ponds at Kaloko-Honokohau National Historical Park

-- -- I I Dissolved Oxygen of KAHO Ponds

I Date 1 -+- Pond 83 tPond 84 +Pond 85 /

I Figure 21: Dissolved oxygen of ponds at Kaloko-Honokohau National Historical Park

Kaloko Pond 83

Date

/ +Tide Height -t Salinity -+Shrimp Density 1

--. I Figure 23: Shrimp density, tide height, and salinity of Pond 83 at KAHO

I I Kaloko Pond 84

Date

/de Height +Salinity tShrimp Density 1 I Figure 24: Shrimp density, tide height, and salintiy of Pond 84 at KAHO I Kaloko Pond 85

29.00 , 1 4000

Date

i+ Tide Height tSalinrty -+Shrimp Density ---

Figure 25: Shrimp Density, tide height, and salinity of Pond 85 at KAKO