histological findings, cadmium bioaccumulation, and ... - BioOne

Journal of Shellfish Research, Vol. 26, No. 4, 1225–1237, 2007.

HISTOLOGICAL FINDINGS, CADMIUM BIOACCUMULATION, AND ISOLATION OF EXPRESSED SEQUENCE TAGS (ESTS) IN CADMIUM-EXPOSED, SPECIFIC PATHOGEN-FREE SHRIMP, LITOPENAEUS VANNAMEI POSTLARVAE

JOHN KEATING,1,2* MARTHA DELANEY,2,3* DAWN MEEHAN-MEOLA,2,3 WILLIAM WARREN,2 ARACELLY ALCIVAR2 AND ACACIA ALCIVAR-WARREN2–4† 1 Pathology Section, Department of Clinical Sciences; 2International Marine Shrimp Environmental Genomics Initiative (IMSEGI); 3Environmental and Comparative Genomics Section, Department of Environmental and Population Health; 4Tufts Center for Conservation Medicine, Cummings School of Veterinary Medicine at Tufts University, North Grafton, Massachusetts 01536 ABSTRACT Cadmium (Cd) is an ubiquitous environmental pollutant of increasing worldwide concern. It has become one of the most hazardous heavy metals in aquatic environments and could threaten aquatic organisms, including marine shrimp. Shrimp are sensitive to Cd and have been found to accumulate it in their bodies in proportion to environmental concentrations. The effects of Cd on the biology and gene expression of the commercially important Litopenaeus vannamei are unknown. The overall hypothesis is that Cd exerts effects on shrimp at both biological and molecular levels. These changes may provide a way to identify genes responsible for toxicity, detoxification and/or tolerance to Cd exposure both acute and chronic. To test the hypothesis, a small-scale pilot project was initiated to obtain baseline information on histological changes associated with Cd treatment and to develop the genomics tools needed to identify genes associated with Cd exposure. The specific objectives of this study were to (1) observe histopathologic changes in control- and Cd-treated postlarvae (PL) during a 48-h period, (2) measure Cd concentrations in Cd-exposed and untreated PLs, and (3) isolate expressed sequence tags (ESTs) from control and Cd-treated PLs for future genomics studies. Specific pathogen-free L. vannamei PLs at stage 42 (PL42, from the Kona Line of the United States Marine Shrimp Farming Program) were used in a waterborne bioassay to determine histological changes in PLs exposed to a range of concentrations of CdCl2 (0, 0.01, 0.1, 1.0, and 10.0 ppm) for 48 h. Results for objective (1) indicated variable response of individual shrimp to Cd exposure at different concentrations. All but 2 animals from 10-ppm group died by 24 h. Histological lesions were limited to the integument, musculature, gills, hepatopancreas, and midgut-hindgut. For objective (2), results showed that the Cd levels in control PLs at 0 h were low and remained relatively low throughout the study. There was a dose- and timedependent relationship of waterborne Cd exposure and accumulation. Results from objective (3) suggested differential gene expression in control- and Cd-treated PLs as reflected by the number of ESTs homologous to genes with different molecular function isolated from approximately 1,100 clones each from the control and Cd-exposed (1 ppm) cDNA libraries. These ESTs contributed to the establishment of an EST database for L. vannamei (ShrimpESTBase). Homology searches of the nucleotide and translated amino acid sequences of ESTs isolated from the control and Cd-treated (1 ppm) cDNA libraries identified a significant number of clones similar to (a) known housekeeping genes and genes involved in immune recognition, signal transduction and effector function, (b) other shrimp ESTs of unknown function, (c) ESTs from other species, (d) predicted, unknown or unnamed proteins from other species, and (e) no homology to any sequence in the GenBank database. Some ESTs (;30%) from the Cdtreated library showed homology to unique sequences representing potential transposable elements. The results provide baseline information on the potential effects of Cd on shrimp health and growth and suggest a complex interaction between environmental conditions, water, feed, stress, and genetic background of PLs and should be further investigated under both laboratory and commercial conditions. Moreover, data suggest L. vannamei may be useful as bioindicators for the condition of their natural environment. KEY WORDS: Litopenaeus vannamei, shrimp, postlarvae stage 42, cadmium bioaccumulation, acute toxicity, histology, ESTs, transposable elements, immune- and stress-related genes

INTRODUCTION

Because of human activities, cadmium (Cd) has become one of the most hazardous heavy metals in aquatic environments and could threaten aquatic organisms, including marine shrimp. Cd is a naturally occurring toxic heavy metal with no known biological function in humans and animals and therefore considered nonessential (FDA 1993, Pinot et al. 2000). Although levels found in nature are believed to be nontoxic, human activities have generated up to 3,700 tons of Cd per year (Pinot et al. 2000 and references therein). Recent studies have indicated that chronic exposure to sublethal levels of Cd can be detrimental to humans (Satarug & Moore 2004). Cd has been *Both authors contributed equally. †Corresponding author. E-mail: [email protected]

shown to be a developmental toxicant in animals, resulting in fetal malformations and other effects, but no conclusive evidence exists in humans (Calabrese & Kenyon 1991, USDHHS 1993, USFDA 1993, USEPA 2000, Zhou et al. 2004). Cd is known to be a carcinogen in animals and suspected to be a human carcinogen of increasing environmental and occupational concern (Mikhailova et al. 1997), it has been classified as a Group B1, probable human carcinogen (USEPA 2000) yet the mechanisms underlying Cd’s carcinogenicity remain unclear (Kasprzak & Bialkowski 2000). Heavy metal cations, including Cd, have the ability to bind to DNA, accumulate within the nuclear chromatin and cause a heritable alteration in the genetic information of the exposed cell(s), leading to transformation of the cell that may ultimately lead to cancer (Kasprzak & Bialkowski 2000, Klaassen et al. 1999, Mikhailova et al. 1997). Targets of Cd include liver, testes, and

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renal epithelial cells with one study concluding that as much as 10% of the general human population may exhibit Cd-dependent renal dysfunction (Bonham et al. 2003 and references therein). In Japan, Cd is believed to cause a condition known as ‘‘Itai-Itai’’ (ouch-ouch) disease in humans; symptoms include nephrotoxicity, hepatotoxicity, nervous system damage, and high frequency of chromatid aberrations (Nordberg 2004, Uetani et al. 2006). Marine shrimp are also sensitive to Cd and have been found to accumulate it in their bodies in proportion to environmental concentrations (Nimmo et al. 1977), however research is limited on Cd effects on shrimp, specifically the Pacific whiteleg Litopenaeus vannamei (Paez-Osuna & Tron-Mayen 1995, Paez-Osuna & Ruiz-Fernandez 1995, Alcivar-Warren et al. 1999, Alcivar-Warren et al. 2001a, Alcivar-Warren et al. 2001b, Alcivar-Warren et al. 2004, Alcivar-Warren & Meehan 2001, Wu & Chen 2004, Wu & Chen 2005a, Wu & Chen 2005b), particularly considering that they may cause indirect adverse effects on human beings through the food chain and even spread these effects globally through commercial importation and exportation (Wu & Chen 2005b). Litopenaeus vannamei is an important species in commercial fisheries along its natural range from northern Mexico to northern Peru and is today the principle species farmed worldwide, including the United States and Asia. Little is known about the relationship between Cd and its effects on the biology, growth, and gene expression in L. vannamei (Vanegas et al. 1997, Wu & Chen 2005b, Wu et al. 2005, Frias-Espericueta et al. 2001). To better understand these relationships, and gain insight on the cellular and molecular mechanisms of Cd toxicity, carcinogenicity and/or tolerance, modern genomics tools need to be developed. These tools include (a) construction of a high-density linkage map for shrimp, (b) cloning of cDNA libraries to isolate expressed sequence tags (ESTs) from Cd-exposed and unexposed shrimp for marker development, mapping and microarray studies, and (c) production of resource families needed to identify candidate genes or quantitative trait loci (QTLs) associated with Cd bioaccumulation and/or tolerance, toxicity, detoxification, and carcinogenicity in shrimp. Currently, a low-density linkage map for specific pathogen-free (SPF) L. vannamei (ShrimpMap) has been constructed based mostly on simple sequence repeat (SSRs) or microsatellite genetic markers isolated from genomic libraries (Alcivar-Warren et al. 2007a). Efforts are now directed to increase density of the shrimp linkage map using microsatellite-containing ESTs (EST-SSR) markers that would directly sample variation in the transcribed regions of the shrimp genome and enhance their utility in comparative genomics, as well as for conservation and exploitation of shrimp genetic resources. To date, no ESTs from Cd-exposed L. vannamei have been reported. Histology can be used as an additional tool to monitor the impact of aquatic pollutants on shrimp in their natural environments and aquaculture (Nimmo et al. 1975, Nimmo et al. 1977, Vogt 1987 and references therein). For instance, the midgut gland of Penaeus monodon was found heavily damaged after exposure to 1 ppm of ‘‘Perfekthion’’ (a dimethoate-based insecticide). Nimmo et al. (1975) observed that P. duorarum developed crystalloids in hepatopancreatic nuclei after exposure to low levels of Arochlor R-1254. Nimmo et al. (1977) observed lesions in gills of P. duorarum exposed to 1–5 ppm of Cd. As suggested by Vogt (1987), structural alterations repre-

sent a highly sensitive overall response of shrimp to the synergistic effect of their nutritional and physiological condition and the quality of the water. Moreover, Johnson & Bergmann (1984) and Moore (1985) suggested that the impact of pollutants in an organism should be realized as perturbations at different levels of functional complexity, by measuring the severity of a toxicant at different levels: molecular, cellular, tissue, organ, individual, or population level; and histological diagnosis should be used as a highly sensitive methods to show the response of an animal to the impact of a toxicant. The specific goals of this study are to (1) observe histological changes in control- and Cd-treated PLs during a 48 h period, (2) measure Cd concentrations in Cd-exposed and untreated PL42s, and (3) isolate expressed sequence tags (ESTs) from control and Cd-treated PLs for future genomics studies. We report here baseline information on histological changes, Cd bioaccumulation, and expressed genes identified in control and Cd-exposed SPF L. vannamei postlarvae. MATERIALS AND METHODS Experimental Animals and Laboratory Facilities

Specific pathogen-free (SPF) L. vannamei postlarvae (PL) from the United States Marine Shrimp Farming Program (USMSFP) (Carr et al. 1997, Wolfus et al. 1997, Moss et al. 1999, Xu et al. 2003, Hennig et al. 2004) were used in this study. PLs originated from a batch of 10 families of the USMSFP’s Research (Kona) Line (Xu et al. 2003, Hennig et al. 2004). PLs at developmental stage 31 (PL31) were shipped from The Oceanic Institute (OI, Kona, HI) to Tufts Cummings School of Veterinary Medicine (TCSVM, North Grafton, MA) and maintained at the wet laboratory at 25°C under a 12:12 h lightdark photoperiod. The wet laboratory is equipped with three Aquaneering Zebrafish 9.0-L tanks (Allentown Caging Equipment Company, Inc., Allentown, NJ) filled with Instant Ocean (Premium Aquatics, Inc., Indianapolis, IN) artificial seawater at salinity range of 23–26 ppt. Water quality was maintained at an average pH of 8.0 (±0.4) and ammonia levels within normal limits (15 foci or too numerous to count.) The histological differences in organs/tissues of control PLs and Cd-treated PLs were analyzed by a board-certified veterinary pathologist (JK). Measurement of Cadmium Concentrations in Shrimp Postlarvae

Cadmium concentrations were determined by Environmental Testing and Research Laboratories (Leominster, MA) using acid/peroxide digestion and Inductively Coupled Plasma Mass Spectroscopy (ICP/MS). Two animals per replicate per time point (at 12, 24, and 48 h, total of 6 animals) were collected, rinsed, and stored at –20°C until Cd analysis was performed. Twenty PLs from time 0 were also sent for analysis. The replicate samples for each time point were pooled and whole body PLs were homogenized before analysis was performed. Cadmium concentrations were also analyzed in samples of Ziegler Aqua feed used during acclimation period and in stock solutions of 1,000 ppm and 100 ppm CdCl2. The stock solutions were maintained at room temperature and tested a year after the study was completed. cDNA Library Construction

Two cDNA libraries were constructed by Amplicon Express, Inc. (Pullman, WA). The control library was prepared using PLs from the pool of 71 individuals collected in liquid nitrogen and stored at –80°C before experiment began (0 h). The Cdtreated library was prepared using PLs from the 1.00 ppm 24 h group based on the histological findings (see Results section below). A total of six animals per replicate per time point (12, 24, and 48 h) were collected, except for the 10-ppm treatment group, as they were all dead by 48 h. During collection, live shrimp were taken out of their respective treatment tank, filtered in a fine mesh net, rinsed 3 times in clean artificial seawater at reduced salinity (15 ppt) and placed immediately in liquid nitrogen until they were placed at –80°C for long-term storage. The time for the collection process from start to finish ranged from 15–30 sec, except for 12-h samples. The 12-h samples were not candidates for cDNA library construction because of uncertainty of time elapsed of tissue handling and therefore RNA quality. The cDNA libraries were constructed a

year later. Samples were shipped overnight in dry ice to Amplicon Express, Inc. Amplicon Express staff was responsible for further sample preparation including separation of head (cephalothorax) from tail and immediate placement of the cephalothorax into their proprietary RNA isolation buffer. The mRNA purification and cDNA library cloning protocols followed standard procedures of Amplicon Express. DNA Sequencing and BLAST Analysis

An aliquot of the cDNA libraries was shipped from Amplicon Express to Agencourt, Inc. (Beverly, MA) for sequencing. Approximately 1,100 positive recombinant clones each from the control and Cd-treated libraries were sequenced. Sequence comparisons were performed by querying the sequences against the nonredundant and EST databases of Blastn and Blastx in GenBank (http://www.ncbi.nlm.nih.gov/Genbank/index.html, NCBI-GenBank Flat file Release 156.0). Output files were created for easy access to individual sequence information and automatic programming established in the shrimp EST database so that it will continuously update new homologous genes and other known sequences. The statistical significance of DNA sequence homologies was based on reported cut-off values of E value [E()] < 0.001 (Pearson 2000). EST sequences were stored in Tuft’s Shrimp EST Database (ShrimpESTbase) for future development of polymorphic markers for linkage mapping (Delaney et al. 2007). ESTs were grouped by molecular function/biological processes and summarized accordingly. RESULTS AND DISCUSSIONS

There were survivors at all treatments up to 24 h, however there were only 2 survivors from the 10-ppm treatment at that time. By 36 h, a significant number of animals had died in all treatments. At the end of the experiment (48 h) and after the last sampling sequence (RNA, histology, and Cd whole body), there were 32, 24, 24, 32, and 0 remaining shrimp in control, 0.01, 0.1, 1.0, and 10.0-ppm treatments, respectively. It is possible that complex interaction between insufficient environmental conditions, water, feed, stress, size differences, and genetic background of PLs contributed to these observations. Wu & Chen (2004) reported LC50 at 48 h of 1.30 ppm Cd in L. vannamei, yet we did not observe any difference in PL42 survival in control and 1 ppm Cd-treated groups, suggesting that SPF PLs from the Kona line of the USMSFP may respond differently to Cd exposure than other L. vannamei stocks. Considering the limited number of samples, no conclusions regarding survivorship or growth after Cd exposure can be made from this study. A similar bioassay should be repeated for a longer period of time under laboratory and commercial settings to examine the relationship of Cd levels and shrimp biology and growth performance. Histological Changes in Control- and Cd-treated Postlarvae in a 48-h Period

Histological analysis indicated variable response of individual shrimp to Cd exposure at different concentrations. Lesions were limited to the integument (oral region and appendages were analyzed), musculature, gills, hepatopancreas, and midgut-hindgut (Table 1). Examples of lesions observed in control PLs and PLs exposed to 10 ppm are shown in Figure 1A to F.

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Figure 1. Histological observations in control and Cd-treated SPF L. vannamei postlarvae. 1A: Normal gills from control L. vannamei PL42 (0 ppm, 0 h). 1B: Segmentally affected gill from Cd-treated PL42 (10.0 ppm, 48 h) demonstrating distention of filaments by hemocytes, and lamellation and melanization of the epithelium (3400). 1C: Normal midgut, control, 0 ppm, 0 h (3400). 1D: Marked segmental midgut inflammation with expansion of the lamina propria by cellular infiltrate and associated ulceration and luminal debris from Cd-treated PL42 (10.0 ppm, 48 h). 1E: Normal hepatopancreas, 10.0 ppm, 24 h (3400). 1F: Focal hepatocellular necrosis, as characterized by disruption and fragmentation of tubules and necrosis of epithelial cells (10.0 ppm, 24 h).

None were associated with any evident infectious etiology (i.e., viruses, bacteria, fungi, and protozoa). Detailed histological observations are presented later. Lesions present in control shrimp were limited to gill crusting, melanosis, and hypercellularity, whereas Cd-treated PLs exhibited lesions of the gills and other sites (see Table 1). Gills

Histological changes in the gills fell into two categories. More frequent was degeneration and necrosis of superficial/ epithelial layers (Fig. 1A, 1B). Affected lamellae were lined or

replaced by crusts (sloughing) of flattened cells that exhibited cytoplasmic eosinophilia, brown discoloration (melanization) and loss of cellular detail. Some gills exhibited more diffuse hypercellularity, presumably because of accumulation of hemocytes with resulting distention of hemolymphatic spaces. The hypercellularity was evident in the primary/secondary filaments and central axis. The gill lesions seen here were similar to those reported in juvenile and subadult P. duorarum by Nimmo et al. (1977) using a waterborne assay for 30 days. An ultrastructural study of lesions in gills of Cd-treated P. duorarum revealed changes in mitochondria morphology (Couch 1977).

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Integument

Some appendages (maxillipeds and pereiopods) and paragnaths exhibited segmental cuticular degeneration and necrosis. Similar to the described gill lesions, these were characterized by cuticular disruption and replacement by crusts of slightly granular, basophilic to grey-brown pigmented material with underlying piled-up and flattened epithelial cells, hemocytes, and cellular debris. Musculature

Few shrimp exhibited angular, wedge-shaped foci of myofiber degeneration with fibrosis. The lesions typically were found along the distal tail in PLs of 0.1, 1.0 and 10.0 ppm groups. Midgut and Hindgut

There were infrequent lesions involving varying segments of the midgut, from the hepatopancreas to the posterior midgut cecum. Lesions varied from mild (necrosis and sloughing of scattered epithelial cells) to severe (segmental ulceration with expansion of the lamina propria by hemocytes and distention of gut lumen by sloughed cells) (Fig. 1C, 1D). Other studies with cultured shrimp indicated that the midgut gland (central site of metabolism) is a sensitive organ that could be used to monitor changes in water conditions. For instance, the midgut gland was heavily damaged after exposure of P. monodon to 1 ppm of dimethoate-based insecticide well before significant changes in the behavior of the shrimp could be observed (Vogt 1987).

groups, and their severity was unrelated to the Cd treatment concentration. It is possible that the gill and cutaneous lesions seen in this experiment were related to other factors such as water quality or stress as a result of crowding or starvation. The total number of lesions observed in all treatment groups over 48 h is shown in Figure 2A. Close data analysis shows that PLs sampled later during the experiment (24 and 48 h) had more lesions in the gills than the PLs sampled before the experiment began (0 h) and at 12 h (not shown). The distribution of lesions over 48 h by tissue and Cd treatment is shown in Figure 2B. Data indicates that lesions in the integument (oral region and appendages), hepatopancreas, midgut, and hindgut were only seen in treated PLs and were not found in the control animals. Though there is an ultrastructural study of gill lesions in Cdexposed P. duorarum (Couch 1977) and the impact of Cd on the structure of gills and epipodites of P. japonicus has been described (Soegianto et al. 1999), this is the first report of histological observations for control and Cd-exposed SPF L. vannamei PL42 under laboratory conditions, and with further analysis under more controlled laboratory and commercial

Hepatopancreas

Rare shrimp exhibited isolated foci of necrosis (Fig. 1E, 1F). No report on histological lesions have been described for marine shrimp hepatopancreas even though it is one of the tissues known to accumulate the highest levels of Cd in P. durarum (Nimmo et al. 1977). Hemolymphatic System

Only one shrimp (10 ppm group) exhibited very prominent mural thickening of the cranial-dorsal hepatopancreatic vessel by hemocytes. Hematopoietic Tissue

No histological changes were noted (including necrosis or degeneration). In general, there was no consistent pattern of histological lesions present that could be reliably attributed to Cd exposure. This may be because of either the small number of animals analyzed to date or genetic differences of the pooled offspring of the 10 families from the Kona Line used for this study. In addition, the small size of the shrimp and sagittal sectioning resulted in inconsistent types and amounts of tissue present on each slide. Examination of multiple slides from a tissue block sometimes reduced this complication, and large and prominent structures such as the integument, gills, hepatopancreas, gut, cardiovascular system, nervous system, and hematopoietic tissue were considered to be adequately evaluated. In some cases, however, some tissues were not present for evaluation indicated as (n/a) in Table 1. Histological lesions in the gills and integument were present and sometimes prominent in both treatment and control

Figure 2. Histological lesions observed in L. vannamei postlarvae stage 42 before and after treatment with various concentrations of cadmium. A$ total lesions observed in all treatment groups over 48 h. B$ number of lesions in different tissues over 48 h. Data indicate that lesions in the integument (oral region and appendages), hepatopancreas, midgut, and hindgut were only seen in treated PLs and were not found in the control animals.


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settings, Cd-induced histopathologic changes will be better understood. Cadmium Concentrations in Control and Cd-treated Postlarvae

The Cd levels in control PL42 from the Kona Line were low at 0 h (0.165 ppm) and remained low throughout the study, with levels reaching 0.350 ppm by 48 h after Cd exposure. Low metal levels were also reported in SPF adult L. vannamei (AlcivarWarren & Meehan 2001) maintained at different grow-out systems (Moss et al. 2001). Cadmium concentrations in L. vannamei PLs exposed to various CdCl2 concentrations during time intervals are shown in Table 2 and Figure 3A. The percentage of change from controls is shown in Figure 3B. Results indicated that there is a dose-dependent as well as a time-dependent relationship of water-borne Cd exposure and accumulation. Cd treatments of 1.0 ppm and 10.0 ppm caused the most accumulation of Cd (Table 2). Linear regression analysis showed a significant correlation (r2 ¼ 0.924, P < 0.0001) between Cd treatment concentrations and Cd concentrations found in PLs whole body (Fig. 4). It is expected that exposure of shrimp to low Cd levels over short or extended period of time will also accumulate at various rates in different tissues. Penaeus duorarum accumulated Cd after 96 h at concentrations proportionate to those present in seawater and this accumulation was tissue-specific, with the hepatopancreas containing the highest levels, followed by exoskeleton, muscle and serum. In P. duorarum muscle, the maximum Cd concentration measured after exposure to seawater containing 0, 0.079, 0.182, 0.307, 0.586, 0.866, and 1.285 ppm Cd for 30 days in a flow-through bioassay was 0.4, 3.8, 10.4, 17.0, 19.4, 30.1, and 30.5 ppm Cd, respectively (Nimmo et al. 1977). The Cd levels accumulated in whole, cultured SPF L. vannamei PL42 using artificial seawater (Table 3) are lower

Figure 3. A. Cadmium concentrations in pooled whole bodies of SPF L. vannamei PL42 from the Kona Line of the USMSFP (control and CdCl2exposed for up to 48 h) determined by ICP/MS. B. Percentage change in cadmium concentrations in CdCl2–exposed L. vannamei postalarvae over control. See Materials and Methods for details.

TABLE 2.

Cadmium concentrations in control (0) and Cd-exposed SPF Litopenaeus vannamei postlarvae stage 42 from the Kona Line of the USMSFP.a. Cadmium Treatment (ppm)

Exposure Time (hours)

Cadmium Concentration (ppm)a

0 0 0.01 0.1 1.0 10.0 0 0.01 0.1 1.0 10.0 0 0.01 0.1 1.0

0 12 12 12 12 12 24 24 24 24 24 48 48 48 48

0.165 0.223 0.353 0.881 4.580 13.330 0.175 0.780 1.370 6.100 15.600 0.350 0.350 2.230 15.820

a

Cadmium concentrations correspond to a pooled sample of six whole L. vannamei PL42 (2 Pls per replicate). No information available for 10.00 ppm Cd treatment because all animals were dead by 48 h.

than those accumulated in muscle of wild P. duorarum from seawater (Nimmo et al. 1977) and may reflect either different environmental conditions (salinity, temperature, etc.), species genetic differences, or the effect of many generations of selective breeding in culture settings. The PL42 used in this study were from the Kona Line, which, in addition to consistently being highly susceptible to Taura Syndrome Virus (TSV); White Spot Syndrome Virus (WSSV); and other pathogens, also perform well under intensive growing conditions and are distributed to local shrimp farmers for grow out purposes (Henning et al. 2004). It is unclear if PLs from other SPF lines of the USMSFP would also accumulate Cd as the Kona Line. Considering the sample size and environmental variables that may have affected the outcome of our study, the results should be taken with caution. Considering that the Kona Line contributed to the development of the High Growth Line and the new TSVresistant line of the USMSFP, and the TSV-resistant line also performs well under intensive raceways conditions, and these lines originated from different geographic regions, it will be important to determine the LC50 for Cd in PLs from all the SPF lines in the USMSFP germplasm. It is also possible that accumulation of Cd is genetically determined in L. vannamei, and Cd may act as a positive endocrine regulator stimulating growth of SPF shrimp under some environmental conditions.

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Figure 4. Simple Linear Regression plot showing a significant correlation (r2 $ 0.924, P < 0.0001) between cadmium treatment concentration and concentrations found in whole body L. vannamei PL42 exposed to cadmium for 24 h. Note: the 48 h values were excluded, see Materials and Methods and Results sections for details.

The association between Cd bioaccumulation and growth performance should be rigorously tested under laboratory and commercial settings. To date, no information is available about the association of heavy metals with viral disease susceptibility. Liu et al. (2006) reported that VP9, a full-length protein of WSSV, binds with both Zn2 + and Cd2+. VP9 was found to adopt a similar fold as the DNA binding domain of the papillomavirus E2 protein suggesting that VP9 might be involved in WSSV expression. Research is needed on the potential association of acute and chronic levels of divalent metal ions and WSSV expression and its transcriptional regulation. Moreover, considering that there were no differences in survival of Kona PL42 individuals after 48 h exposure to Cd concentrations of 0.01, 0.10, and 1.00 ppm, a range of concentrations known to cause differences in mortality rates in other shrimp species, it is tempting to speculate a role for Cd as a positive endocrine regulator of high growth of the Kona line. Research is needed to determine the LC50 and bioaccumulation of Cd at different stages of shrimp development using PLs from genetically different shrimp stocks such as the TSV-resistant and High Growth lines maintained by the USMSFP. Results from this study show that SPF L. vannamei PL42 from the Kona Line are capable of accumulating Cd well above maximum levels (0.5 ppm) accepted by the European Union, FAO and WHO (Joint FAO/WHO Expert Committee on Food Additives 2006) and approaching FDA standards (3.0 ppm) after low level Cd exposure (Table 3). Cadmium bioaccumulation has been studied in various crustaceans including crabs, lobsters, and shrimp. The hepatopancreas, midgut, gills, and exoskeleton have been found to sequester Cd in crustacean whereas the muscle tissue and serum are not considered sites of significant accumulation (Paez-Osuna & Tron-Mayen 1995, Nimmo et al. 1977). In penaeid shrimp species, the hepatopan-

creas is the main organ of Cd accumulation, differing by one order of magnitude when compared with exoskeleton and three orders of magnitude when compared with serum (Nimmo et al. 1977). In wild Fenneropenaeus merguiensis, higher Cd levels were found in cephalotorax than in tail muscle (Panutrakul et al. 2007). Cd levels were also found 32– 33 higher in cephalothorax of wild L. vannamei of Ecuador and El Salvador (Alcivar-Warren et al. 2001a) and P. monodon of Philippines (Alcivar-Warren et al. 1999) when compared with levels in tail muscle with and without exoskeleton. Considering the potential for adverse health effects from Cd exposure (Nordberg 2004), even at low Cd levels, molecular epidemiology studies should be performed to assess potential risk to shrimp and human health caused Cd bioaccumulation and provide basis for preventive action. It is possible that long-term exposure to heavy metals contamination alters both gene expression and allele frequencies in wild and cultured shrimp populations. A continued decline in genetic diversity of fingernail clams was found associated to pollution (Sloss et al. 1998). Changes in allele frequencies of two genetic markers (phosphoglucomutase and glucose phosphate isomerase) and levels of metallothionein protein were observed in clams after heavy metal challenges (reviewed in Camara et al. 2005). Genetic diversity and heterozygosity of marine organisms seems to be related to heterogeneity and environmental stress (Nevo et al. 1983, Nevo et al. 1986). These researchers found that marine gastropods with high genetic diversity were more resistant to organic and inorganic pollutants than their counterparts, suggesting that fitness is positively correlated with heterozygosity. Differential survivorship of shrimp allozyme genotypes specific for Cd, Hg, and their interaction, were also reported (Ben-Schlomo & Nevo 1988), suggesting that rapid and precise changes can follow the effects of pollution. Moreover, exposure to Cd leads to a wide reorganization of S. cerevisiae transcriptome and proteome, resulting in a significant increase in glutathione synthesis regulated by transcriptional activators of the sulfur metabolism enzymes (Baudouin-Cornu & Labarre 2006) and a complex effect on cell cycle regulation (Yen et al. 2005, Othumpangat et al. 2005). However, different pollutants are expected to affect different genotypes, depending on the population’s structure and organization of the genome. Genomics tools such as highly saturated genetic linkage maps and microarrays will facilitate identification of genes or QTLs associated with Cd bioaccumulation and Cd tolerance (Delaney et al. 2007). Cd Stock Solutions and Shrimp Feed

The Cd levels found in the 100 ppm and 1,000 ppm CdCl2 stock solutions used to prepare dilutions in the bioassay were 102.5 ppm and 891.0 ppm, respectively. These samples were submitted over a year after the solutions were made, possibly explaining the discrepancy in the Cd concentrations. The Ziegler Aqua shrimp feed (#1 & #2) used in this waterborne bioassay had Cd levels of 0.780 ppm and 0.290 ppm, respectively. These levels are relatively high. Shrimp feed, in addition to the water, may be a vehicle for Cd accumulation and needs further research. As PLs in our study were not fed during the experiment, this could be a potential reason why our controls had low levels of Cd bioaccumulation. Availability of a semipurified shrimp diet would be most valuable for future Cd research.


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TABLE 3. –1

Cadmium concentrations (mg g ) measured in whole, cultured, specific pathogen-free Litopenaeus vannamei postlarvae stage 42 (PL42) treated with Cd in a waterborne assay using artificial seawater, and comparison with the Cd levels reported from LC50 studies, found in other penaeid shrimp species, and the internationally recommended safety limit.a Cadmium safety limits are 2.00, 3.00, and 0.50 mg/g (ppm) based on published international standardsa–c. Species (Reference; Objective) Bioaccumulation studies L. vannamei (this study, to measure Cd concentrations in control untreated PLs and observe histopathologic changes for 48 h) L. vannamei (this study, to measure Cd concentrations in Cd-exposed and control untreated PLs and observe histopathologic changes), PLs were exposed to 0.01, 0.10, 1.00 ppm Cd for 12–48 h

L. vannamei (this study, whole PL42 exposed to 10.0 ppm Cd for 24h) L. vannamei (Wu & Chen 2005b, studied Cd accumulation in hepatopancreas, gills and muscle) P. durarum (Nimmo et al. 1977, studied Cd accumulation in muscle of shrimp exposed to various concentrations of Cd in test water for 30 days in a flow-through bioassay)

LC50 studies Penaeus japonicus (Bambang et al. 1994, studied effect of Cd on survival, tolerance and development)

Penaeus durarum (Nimmo et al. 1977)

L. vannamei (Wu & Chen. 2004)

L. vannamei (Frias-Espericueta et al. 2001) Trace concentrations in wild and cultured shrimp L. vannamei (Alcivar-Warren & Meehan 2001, measured Cd in SPF broodstock kept in recirculating, zero discharge system) L. vannamei (Alcivar-Warren & Meehan 2001, measured Cd in broodstock kept in a flow-through earthen pond) L. vannamei (Alcivar-Warren et al. 2001b, measured Cd in wild brood stock from El Salvador -kept in an earthen pond in Ecuador for breeding purposes-) L. vannamei (Alcivar-Warren et al. 2001b, measured Cd in wild brood stock from Ecuador kept in earthen pond in Ecuador, for breeding purposes) Ocean and pink shrimp (NMFS, survey of wild shrimp) P. monodon (Alcivar-Warren et al. 1999, monitored heavy metals in wild shrimp from four geographic regions of Philippines) P. monodon (Alcivar-Warren et al. 1999, monitored heavy metals in cultured shrimp from SEAFDEC, Philippines) Fenneropenaeus merguiensis a

Concentration of Cd Detected, mg/g or ppm (shrimp Part, Country) 0.17–0.35 (whole, pooled, cultured, PL42, control no Cd exposure, Kona, USA) 0.35–0.78* (whole, pooled, cultured, PL42, 0.01ppm Cd-exposed, Kona, USA) 0.83*–2.23* (whole, pooled PL42, 0.10 ppm Cd-exposed, Kona, USA) 4.58*–15.82* (whole, pooled PL42, 1.00 ppm Cd-exposed, Kona, USA) 13.30*–15.60* (whole, pooled PL42, 10.00 ppm Cd-exposed, Kona, USA) 0.10 ppm (hepatopancreas, juveniles, 0.10 ppm Cd-exposed, Taiwan) 3.80* (muscle, wild, from seawater with 0.079 ppm Cd) 10.40* (muscle, wild, from seawater with 0.182 ppm Cd) 17.00* (muscle, wild, from seawater with 0.307 ppm Cd) 19.40* (muscle, wild, from seawater with 0.586 ppm Cd) 30.10* (muscle, wild, from seawater with 0.866 ppm Cd) Nauplii: 48h LC50: 0.124 Zoea 96h LC50: 0.010–0.030 Postlarvae 96 h LC50: 0.200–3.500* Juveniles 96 h LC50: 5.500* Juvenile/subadult, 96 h LC50: 5.000* Juveniles/subadults, 21–25 days LC50: 1.000* Adult, 96 h LC50: 3.500* Adult, 30-day LC50: 0.720* Subadult/adult: LC50: 24h ¼ 2.58* 48 h ¼ 1.30* 72 h ¼ 1.14* 96 h ¼ 1.07* Postlarvae: LC50: 96h ¼ 2.49 0.100–0.200 (whole adult, cultured, High Growth line USMSFP, USA) 0.000–0.030 (whole adult, cultured, High Growth line USMSFP, USA) 0.310–1.070* (whole adult, wild, El Salvador)

0.090–0.900* (whole adult, wild, San Pablo, Ecuador)

0.100–0.200 (whole pooled, wild fishery resource, USA) 0.006–1.000* (whole, adult, wild, from Capiz, Negros Occidental, Quezon, and Palawan, Philippines). Shrimp from Capiz had highest Cd levels. 0.034–0.248 (whole, subadult/adult, cultured F1-F3, SEAFDEC, Philippines) 2.27* (wild, cephalothorax, Chantaburi province, Thailandd,e)

Australia and New Zealand Food Authority Amendment No. 53. (2000). US Food and Drug Administration (1993, 2001). c FAO. Report of the Codex Committee on Food Additives and Contaminants. Draft Guideline level for Cadmium in Food (http://www.fao.org/ docrep/meeting/005/x7137e/x7137e20.htm). Joint FAO/WHO Expert Committee on Food Additives (2006). * indicate maximum concentration, higher than safety limit for at least one of the agencies listed. d From Panutrakul et al. (2007). eCephalothorax and tail muscle from wild shrimp of Chantaburi and Trat provinces were tested. b

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Expressed Sequence Tags From cDNA Libraries of Control and Cd-treated Postlarvae

A total of 2,684 recombinant clones from the two cDNA libraries were sequenced. After vector trimming and quality assurance (e.g., deletion of clones with less than 50 nucleotides, too many Ns, etc.), ;2,300 ESTs were useful for further analysis. Homology searches of the nucleotides and translated amino acid sequences of ESTs isolated from the control and Cdtreated (1 ppm) cDNA libraries identified a significant number of clones similar to (a) known genes, (b) other shrimp ESTs of unknown function, (c) ESTs from other species, (d) predicted, unknown or unnamed proteins from other species, and (e) no homology to any sequence in the GenBank database. The known genes in the control cDNA library included genes with different cellular function including: mitochondrial DNA-encoded subunits (mostly 16s rRNA and a few COI, COII, COIII, ATPase, NADH1, and NADH6); 40S and 60S ribosomal proteins and others involved in the translational machinery (S4, S8, S9, L5, L6, L10, L11, L13A, L21, L26, L27, L34, L37, elongation factor 1-gamma); cytoskeleton proteins (actin, troponin, tropmyosin, myosin heavy chain, sarcoplasmic calcium-binding protein, and tubulin beta-1 chain); cell wall proteins (galactomannoproteins, keratin-associated proteins, calcified cuticle protein, chitinase, partial TTN for titin); cell cycle related proteins (stromal cell derived factor 2, transcription factor AP2, S-phase response); vision genes (opsin, rhodopsin), cell-signaling molecules (cyclophillin, yellow head virus receptor protein, laminin receptor, receptor for activated protein kinase C-like, TonB-dependent receptors, wound response gene, peritrophin A, motifs of U88 protein of human herpesvirus 6, antiviral proteins (hemocyanin, crustacyanin, PMAV); and allergens (Pen a 1, arginine kinase, tropomyosin), among others. A large number of ESTs had no similarity to any other sequences in the database. The known genes from the Cd-treated (1 ppm) cDNA library included (a) housekeeping genes such as nuclear DNA-encoded subunits of oxidative phosphorylation; mitochondrial DNA subunits (16s rRNA, COI, COII, COIII, NADH2, NADH3); ribosomal proteins and other genes involved in the translational machinery (S3A, S9, S12, S18, S19, L1p/L10e, L6, L7a, L9, L21, L22(L17a), L23, L26, L27, L34, L37a, internal transcribed spacer 2, elongation factor 1, translation initiation factor 3 subunit 6, acidic ribosomal proteins 1 and 2, 18s rRNA); (b) cytoskeleton proteins (actin, troponin I, tubulin beta-2, myosin heavy chain, I-connectin); (c) metal- and stress-related proteins (iron ion binding proteins, metal ion transporter, heat-induced protein, ubiquitin, thioredoxin), (d) immune response genes including antibacterial peptides (crustacyanin-C1), antiviral genes (hemocyanin), and allergen genes (arginine kinase); (e) cuticle proteins (BCS-1, keratin-associated protein, calcified cuticle protein CP14.1, carcinin-like protein); (f) cell signaling molecules (high mobility protein, sarcoplasmic calcium-binding protein, cyclophillin RNA-interacting protein, PkB-like, adipose enhancer binding protein, zinc finger protein, profilin); and (g) CG6770 orphan gene, among others. Only a few ESTs had no homology to any sequence in the Genebank database. It is possible that differential expression of some of the housekeeping, and potential growth- and development-related genes (such as those involved in the mitochondrial oxidative phosphorylation and translational regulation pathways) identified here in

Kona PLs may relate to Cd accumulation stress. A potential association between susceptibility to viral diseases and high growth performance was also observed in some studies using shrimp from the Kona line (Alcivar-Warren et al. 1997, Moss et al. 1999, Moss et al. 2001, Moss et al. 2002). This is the first report of genes associated with Cd exposure in SPF L. vannamei postlarvae and some of the metal- and stress-related genes reported here are known to be induced even with minute amounts of Cd (Wiegant et al. 1997) or a nonlethal dose of Cd (Yamada & Koizumi 2002). In other species, Cd activates gene transcription through signal transduction pathways, mediated by protein kinase C, cAMP-dependent protein kinase, or calmodulin (Bhattacharyya et al. 2000). In human cells, stress-related genes such as those coding for metallothioneins and heat shock proteins, antioxidant genes, and ubiquitin pathway genes have been identified (Yamada & Koizumi 2002). In yeast cells, mutants deficient in specific ubiquitin-conjugating enzymes and the proteasome are hypersensitive to Cd, suggesting that Cd resistance is mediated in part by degradation of abnormal proteins (Jungmann et al. 1993). Metallothioneins, an intracellular protein known to protect against Cd toxicity (Klaassen et al. 1999) and induced after transient exposure of Cd (Brulle et al. 2007), was not identified in any of the EST sequences from the Cd-treated (1 ppm) cDNA library. The translation initiation factor-1 d subunit, a Cd-responsive protooncogene with oncogenic potential (Joseph et al. 2001), was not identified in these EST sequences. Moreover, the eukaryotic translation initiation factor 4E gene, a cellular target for toxicity and death caused by exposure to cadmium chloride (Othumpangat et al. 2005) was also not identified in our ESTs from the cDNA library of Cd-treated PLs and this may be because of many variables including source of RNA used to clone the cDNA library (i.e. tissue [exoskeleton]) and developmental stage, time of exposure, shrimp line used, etc. Considering that differences in molt frequency and molt cycle duration are associated with growth performance of the Kona line (Moss et al. 2002) more research is needed to understand the mechanisms by which Cd affects growth and early development in PLs from disease susceptible (Kona) and disease resistant (TSV-R) lines from the USMSFP. In addition to known and hypothetical genes, a large number of ESTs (;30%) from the Cd-treated cDNA library contained portions of a motif (of up to ;137–150 bp) with no similarity to any sequence in the GenBank database. Some clones contained sequences similar to nucleotides 1–137 of a human sequence (GenBank accession #BC063380) and/or portions of the cloning vector pDNR-Lib (approx. 100–220 bp) followed by either partial sequences of known genes such as 28S rRNA, chloramphenicol resistant gene, orphan gene, 16s rRNA, ribosomal protein subunits or sequences of unknown function from plant and other species. Some of the ESTs also contained portions (;30–80 nt) of the PP-CAT gene (accession #BAA03718). It is possible that these sequence rearrangements are caused by either cloning artifacts or expression of transposable elements. Sequence rearrangements indicating active expression of nonLTR retrotransposons (reverse transcriptase) and other transposable elements have been observed in L. vannamei sequences isolated from either genomic libraries or cDNA libraries (of White Spot Syndrome Virus-and Taura Syndrome Virus-challenged shrimp), which were cloned into vectors different from the one used in this study (Alcivar-Warren

HISTOLOGICAL FINDINGS AND CADMIUM BIOACCUMULATION IN SHRIMP et al. 2006, Alcivar-Warren et al. 2007b, Garcia & AlcivarWarren 2007, Xu & Alcivar-Warren, unpublished results). Genetic rearrangements putatively caused by retrotransposons were also identified in L. stylirostris shrimp 24 h after infection with infectious hypodermal and hematopoietic necrotic virus (IHHNV), suggesting that stress induces expression of transposable elements (Hizer et al. 2002, Malfavon-Borja et al. 2006). The presence of putative sequence rearrangements after Cd exposure of shrimp postlarvae also suggests that Cd stress induces the expression of transposable elements. The ESTs isolated from both the control and Cd-treated cDNA libraries have been added to the Tufts’ shrimp EST database (ShrimpESTbase) for future microarrays and linkage mapping studies (Alcivar-Warren et al. 2007a). Availability of a highly saturated linkage map will facilitate (1) identification of candidate genes or QTLs associated with tolerance/susceptibility to Cd bioaccumulation, toxicity, and detoxification; (2) introgression of the Cd resistance/tolerance trait through marker-assisted selection; and (3) selective breeding of shrimp resistant/tolerant to Cd bioaccumulation, similar to the breeding program for oyster aimed at reducing Cd content in the species (Camara et al. 2005). In summary, the histological, biological, and genomics results presented here provide baseline information to enable testable hypothesis on the potential positive or negative effects of Cd on shrimp health, growth and development. The levels of Cd in Cd-treated PLs are very high and should be repeated

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under more controlled experimental and commercial conditions before final statements are made about potential risk of Cd levels to environmental, animal, or public health. The ESTs add to the ShrimpESTbase and will be useful not only to develop polymorphic markers to increase density of the shrimp linkage map but provide valuable comparative genomic links between L. vannamei and other penaeid species as well as invertebrate and vertebrate genomes. Based on the different genes isolated from unexposed and Cd-exposed PLs, it seems that molecular changes precede cellular changes in cephalotorax of Cd-treated PLs, similar to the effects reported in mouse testis using lowdose Cd (Zhou et al. 2004). ACKNOWLEDGMENTS

This work was partially supported by the National Institute of Health Short Training Grant #T35DK07635 (MD), the United States Department of Agriculture (USDA-CSREES) through a grant to the United States Marine Shrimp Farming Program (A-W); the Department of Environmental and Population Health, TCSMV (A-W); Tufts Center for Conservation Medicine (MD), and the Pathology Section of the Clinical Sciences Department at TCSVM (JK). The authors thank Dr. Zhenkang Xu for initial suggestions on experimental design during the planning phase of this study, Drs. Paramananda Das and George Saperstein for providing comments on the manuscript, and Ms. Janine Stuczko for editing the manuscript.

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