Market integration between wild and farmed species in Spain

ABSTRACT

Market integration occurs when prices among different locations or related goods follow similar patterns over time. Current knowledge on market integration between aquaculture and wild-caught fish is based on a small number of species and markets. Most studies show the existence of market integration between wild and farmed conspecifics, with the clear exception of European seabass and gilthead seabream in Southern European countries. In this study, we investigate whether this lack of market integration between wild and farmed conspecifics for European seabass and gilthead seabream in Southern European countries is specific for these species or is representative for the area. Therefore, we investigate the existence of market integration in Spain between wild and farmed conspecifics for a large variety of different species: turbot (Scophthalmus maximus), sole (Solea spp.), blackspot (red) seabream (Pagellus bogaraveo), Atlantic cod (Gadus morhua), and meagre (Argyrosomus regius).     

Genetic variation in farmed populations of the gilthead sea bream Sparus aurata in Greece using microsatellite DNA markers

Genetic variation in seven reared stocks of gilthead sea bream Sparus aurata, originating from Greek commercial farms, was assessed using five polymorphic microsatellite markers and was compared with that of two natural populations from the Ionian and the Adriatic Seas. The total number of alleles per marker ranged from 11 to 19 alleles, and hatchery samples showed the same levels of observed heterozygosity with samples from the wild but substantially smaller allelic diversity and expected heterozygosity. The global genetic differentiation for the cultivated samples was significant as indicated by F_st analysis, which might indicate random genetic drift and inbreeding events operating in the hatcheries. On the contrary, no significant difference was found between the two wild populations. Population pairwise tests between farmed and wild stocks were also significant, with the exception of one hatchery sample, the Central Greece 1, which was not significantly different from the two wild samples perhaps due to its recent use in aquaculture from wild‐caught animals. The UPGMA tree topology grouped the wild samples together with the Central Greece 1 stock, and showed a clear division between wild and farmed sample sets for the six remaining hatchery samples. Knowledge of the genetic variation in S. aurata cultured populations compared with that in the wild ones is essential for setting up appropriate guidelines for the proper monitoring and management of the stocks either under traditional practices or for the implementation of selective breeding programmes.     

Microsatellite genetic variation in wild and hatchery populations of the sea cucumber (Apostichopus japonicus Selenka) from northern China

Farming of the sea cucumber Apostichopus japonicus (Selenka) started 20 years ago and is still in rapid expansion in China. In order to assess the genetic status of both wild and cultivated stocks of this species, we used eight microsatellite markers to estimate the level of genetic diversity within five hatchery stocks and two wild populations of A. japonicus, and compared the degree of genetic differentiation between them. High levels of polymorphism were observed over all loci. The mean alleles and expected heterozygosities over the seven stocks were 10.4–12.3 and 0.735–0.783 respectively. The results of the microsatellite survey provide no evidence to show that hatchery practice of the sea cucumber in China to date has significantly affected the genetic variability of the cultured stocks. Significant differentiation was found between most pairs of the hatchery stocks and wild populations (F_st range: 0.008–0.036), and no obvious difference was detected between the wild populations (F_st=0.008). The information on the genetic variation and differentiation obtained in this study can be applied for future genetic monitoring of A. japonicus aquaculture stocks and will be useful for future genetic improvement by selective breeding, and for designing suitable management guidelines for these genetic materials.     

Loss of genetic variability in the captive stocks of tambaqui,Colossoma macropomum (Cuvier, 1818), at breeding centres in Brazil,and their divergence from wild populations

The loss of variability in farmed populations and the risks of interactions with wild populations support the need for the genetic monitoring of species farmed throughout the world. In Brazil, the tambaqui is the most widely farmed native fish species. Despite this, there are no data on the pedigree of the farmed stocks, and the potential for interactions with wild populations in the Amazon basin has raised concerns with regard to the genetic variability of these stocks. The present study analysed sequences of the mitochondrial Control Region and 12 microsatellites to characterize the genetic variability of seven historically important commercial tambaqui breeding centres located in four different regions of Brazil, and compared these sequences with those obtained from individuals collected from a wild population. High levels of genetic diversity were found in the wild population, whereas genetic diversity was reduced in both markers in most captive populations, except for the broodstock located near the Amazon River. High F_ST and D_EST indices were recorded between the wild population and most of the captive stocks analysed. The drastic reduction in genetic diversity found in most captive stocks and the difference between these stocks and the wild population may have been the result of the small size of the founding populations and the absence of breeding management. The renewal of the broodstocks and the application of breeding management techniques are recommended. In the Amazon region, in addition, the use of broodstocks that are genetically very different from local wild populations should be avoided.     

Effect of Two Natural Carotenoid Sources in Diets for Gilthead Seabream,Sparus aurata,on Growth and Skin Coloration

To improve the unnatural fade-pigmented skin of cultivated gilthead seabream, Sparus aurata, (if shown) the present study was initiated. The effects of either red bell-pepper (Capsicum annum) meal or carrot (Daucus carota) meal as a natural dietary carotenoid source, on growth and skin coloration of gilthead seabream growers were investigated. A basal/control diet (D1/CTR) was firstly formulated to contain 48% crude protein and 14% lipids, with no added-carotenoids. With this basal diet, two other test diets were similarly prepared and supplemented each with about 40mg/Kg total carotenoids from either red-pepper meal (D2) or carrot meal (D3). In a feeding trial, fish (mean IW, 94.86±0.3g) were fed one of the three diets (D1, D2, D3), in triplicates for each treatment, for 6 weeks in light-blue background PVC tanks supplied with natural seawater flow. Total carotenoids content of skin was determined spectrophotometerically at initiation and end of the experiment. Neither growth nor feed utilization were significantly (P<0.05) affected by the red pepper-added diet (D2) as compared to CTR diet. However, the carrot fed fish recorded the lowest and significant (P<0.05) weight gain (g/fish) and specific growth rate (SGR, %/d) among dietary treatments. There were no considerable (P>0.05) differences in major nutrients composition between fish fed the experimental diets. Total carotenoids content was significantly (P<0.05) increased, in the skin-opercle area, of fish fed the red pepper diet (D2) as compared to initial fish and to either carrot fed fish or CTR fish. Results have suggested that gilthead seabream can effectively bio-absorb natural carotenoid pigments (mainly capsansin and capsorbin) in red-pepper but not in carrot (mainly β-and α-carotene).     

Characterization of β-haemolytic Group B Streptococcus agalactiae in cultured seabream, Sparus auratus L., and wild mullet, Liza klunzingeri (Day), in Kuwait

Streptococcus agalactiae was isolated from cultured gilthead seabream, Sparus auratus L., and diseased wild Klunzinger's mullet, Liza klunzingeri (Day), in Kuwait Bay, Arabian Gulf. Isolates were catalase negative, β‐haemolytic, Gram‐positive cocci and serogroup B. Experimental infectivity trials with mullet and seabream brain isolates in Nile tilapia, Oreochromis niloticus L., caused 100 and 90% mortality, respectively, within 7 days post‐inoculation indicating virulent S. agalactiae as the bacterial pathogen responsible for the epizootic in Kuwait Bay.     

Interference of Listonella anguillarum with potential probiotic microorganisms isolated from farmed gilthead seabream (Sparus aurata, L.)

Four isolates obtained from gilthead seabream have been tested for their adhesion to the skin, gill and intestinal mucus of gilthead seabream, and for their ability to interfere with Listonella anguillarum, an important pathogen of farmed gilthead seabream. The ability to adhere to mucus was higher than 7% for all isolates. Three isolates showed an antagonistic effect against some of the pathogenic strains tested. They were assayed to interfere with the attachment of L. anguillarum to the mucus of gilthead seabream. Only two isolates significantly reduced the adhesion of L. anguillarum to all of the mucus assayed under exclusion, competition and displacement conditions. According to the criteria applied, the isolate Pdp11 was selected and its in vivo probiotic potential was assessed by oral administration followed by challenge with the pathogen L. anguillarum. For the feeding trial, a group of 50 gilthead seabreams received a commercial diet supplemented with lyophilized 10⁸ CFU g^?1 of this isolate for 15 days. An other group of similar characteristics received the non‐supplemented commercial diet. After the challenge, the mortality of the fish receiving the diet supplemented with the potential probiotic was significantly lower than that observed in the groups of fish receiving the non‐supplemented commercial diet.     

Nutritional stimuli of gilthead seabream (Sparus aurata) larvae by dietary fatty acids: effects on larval performance,gene expression and neurogenesis

The concept of nutritional programming raises the interesting possibility of directing specific metabolic pathways or functions in juvenile fish, for example, to improve the use of substitutes to fishmeal and oil, and hence to promote sustainability in aquaculture. The aim of the study was to determine effects of early nutritional stimuli of gilthead seabream larvae and check if nutritional programming of gilthead sea bream is possible between 16 days post hatching (dph) and 26 dph. A trial was conducted to determine the effects of early nutritional stimuli of gilthead seabream larvae. Five experimental microdiets (pellet size <250 μm) were formulated containing five different proportions of a marine lipid source rich in long‐chain polyunsaturated fatty acids (LC‐PUFA) and two vegetable lipid sources rich in linolenic and linoleic acids. The results of this study demonstrate that dietary n‐3 LC‐PUFA levels increased larval growth and survival affecting Δ6 desaturase gen (fads2) expression and retinal neurons density. However, the high mortalities obtained along on‐growing in fish fed low n‐3 LC‐PUFA at 16 dph constrained the feasibility of nutritional programming of gilthead seabream during this late developmental window and needs to be further investigated.     

Debaryomyces hansenii L2‐enriched diet enhances the immunity status,gene expression and intestine functionality in gilthead seabream (Sparus aurata L.)

This study evaluates the effects of the dietary administration of the live yeast Debaryomyces hansenii strain L2 on the immune responses of gilthead seabream for 4 weeks. Cellular immune parameters were measured from serum and head‐kidney leucocytes respectively. The expression levels of immune‐associated genes were quantified by real‐time polymerase chain reaction. In addition, the profile of intestinal microbiota was studied by denaturing gradient gel electrophoresis. The results showed that seabream fed a diet containing D. hansenii had significantly increased cellular immune parameters. The yeast‐supplemented diet up‐regulated the expression of most seabream genes at week 2 and down‐regulated all of them at week 4, except in the head‐kidney. Finally, a reduction in the diversity of the intestinal microbiota was detected in those specimens receiving the yeast‐supplemented diet. These results support the idea that the live yeast D. hansenii strain L2 stimulates the immune system of gilthead seabream.     

AFLP and microsatellites as genetic tags to identify cultured gilthead seabream escapees: data from a simulated floating cage breaking event

Genetic discrimination using DNA fingerprinting is rapidly developing for cultured stock and wild fish populations. Microsatellites and AFLPs are being widely used in aquaculture to assign fish or processed fish products, to their claimed origin, paternity or strain. In the present study, 147 AFLP and 4 microsatellite markers were used as genetic tags in gilthead seabream, Sparus auratus. Specimens from two different hatchery broodstocks (one of Atlantic and one of Mediterranean origin) and wild fishes from a natural population were fingerprinted. Putative offspring from these broodstocks were computer-generated, and the confidence in the parentage assignment of their genetic profiles to the hatchery broodstock assessed. The virtual offspring were then mixed with specimens from a natural population to simulate an accidental escape from a floating cage. The risk of false paternity inclusion was evaluated to test the ability to identify either Atlantic or Mediterranean hatchery offspring among wild fish. The method proved to be reliable, and could therefore be used to forecast the impact of fish farm escapees.     

Vaccination trials on gilthead seabream (Sparus aurata) against Pasteurella piscicida

A comparative study of the efficacy of two vaccine formulations, a whole-cell bacterin (WCB) and a toxoid-enriched whole-cell vaccine (WCEB), against Pasteurella piscicida was conducted by bath immersion in gilthead seabream in order to evaluate the role of the extracellular products (ECP) as protective antigens against this pathogen. With this aim, two strains showing differences in their ECP composition were used to prepare both vaccines. Only the toxoid-enriched vaccine conferred protection against P. piscicida within a 4-week period. The relative percent survival (RPS) acheived with this type of vaccine ranged between 37 and 41 depending on the bacterial strain and dose used in the challenge. Although this protection level is not very high, we consider that it is valuable considering the economic importance of the fish susceptible to pasteurellosis throughout the world. The booster immunization with the WCEB P. piscicida formulation did not increase the protection levels of gilthead seabream. The antibody response in the sera of both immunized fish groups was very low with no correlation between the level of agglutinating antibodies and the protection. In addition, this vaccine did not confer cross-protection against serotypes 01 and 02 of Vibrio anguillarum.     

Eggs of gilthead seabream Sparus aurata L. as a potential enrichment product of Brachionus sp. in the larval rearing of gilthead seabream Sparus aurata L.

The gilthead seabream Sparus aurata L. is a sequential spawner whose reproductive cycle can be manipulated to obtain year-round egg and fry production. Hence, there is an overproduction of eggs in commercial hatcheries that is normally wasted. Two feeding experiments were conducted with the purpose of investigating the potential use of fresh and freeze-dried gilthead seabream eggs for the enrichment of live preys. The first experiment was also designed to study the possibility of using freeze-drying as a conservation process for eggs in excess. Larvae were reared for 20 days after hatching (DAH),on a diet of Brachionus sp. As experimental controls, the commercial enrichment emulsion SUPER HUFA was used in the first experiment, and the microalgae Nannochloropsis sp. in the second. This study demonstrated the potential of gilthead seabream eggs as an enrichment product to be used in the feeding of the first larval stages of Sparus aurata L. The data obtained emphasize the need to have an overall perspective of the nutritional requirements of the organisms in culture.     

Effect of krill phospholipids versus soybean lecithin in microdiets for gilthead seabream (Sparus aurata) larvae on molecular markers of antioxidative metabolism and bone development

The objective of the present study was to compare the effectiveness of dietary marine phospholipids (MPL) obtained from krill and soybean lecithin (SBL) on the rearing performance and development of seabream (Sparus aurata) larvae. Larvae were fed from 16 to 44 day posthatching (dph) five formulated microdiets with three different levels (50, 70 and 90 g kg^–1) of phospholipids (PL) obtained either from an MPL or from a SBL source. Larvae‐fed MPL show a higher survival, stress resistance and growth than those‐fed SBL, regardless the dietary PL level. Overall, the increase in MPL up to 70 g kg^–1 total PL in diet was enough to improve larval gilthead seabream performance, whereas even the highest SBL inclusion level (90 g kg^–1 PL) was not able to provide a similar success in larval growth or survival. Inclusion of SBL markedly increased the peroxidation risk as denoted by the higher TBARs in larvae, as well as a higher expression of CAT, GPX and SOD genes. Moreover, SBL tends to produce larvae with a lower number of mineralized vertebrae and a lower expression of osteocalcin, osteopontin and BMP4 genes. Finally, increasing dietary MPL or SBL lead to a better assimilation of polyunsaturated fatty acids in the larvae, n‐3HUFA (especially 20:5n‐3) or n‐6 fatty acids (especially 18:2n‐6), respectively. In conclusion, MPL had a higher effectiveness in promoting survival, growth and skeletal mineralization of gilthead seabream larvae in comparison with SBL.     

Development of Gene‐derived SNP Markers and Their Application for the Assessment of Genetic Diversity in Wild and Cultured Populations in Sea Cucumber,Apostichopus japonicus

The Apostichopus japonicus is a valuable aquaculture species in China. In this study, 51 single nucleotide polymorphisms (SNPs) were identified from expressed sequence tags of sea cucumber using high‐resolution melting. The average observed heterozygosity (Ho) and expected heterozygosity (He) were 0.2462 and 0.2897, respectively. Thirty‐two of these loci were used for estimating the genetic similarity and variation between the five hatchery stocks from China and two wild stocks from Japan. No significant differences in Ho or He were observed between the wild and hatchery populations. The pairwise Fst (which ranged from 0.0119 to 0.0236) and the genetic identity (which varied from 0.9802 to 0.9915) showed no significant differentiation between the wild and cultured stocks. The analysis of molecular variance indicated the source of variation was at the level of “within the populations.” The information on the genetic variation and differentiation in cultured and wild populations of A. japonicus obtained in this study is useful for setting up suitable guidelines for founding and maintaining of cultured stocks and for future genetic improvement by selective breeding.     

Transmission of lymphocystis disease virus to cultured gilthead seabream,Sparus aurata L., larvae

The transmission of lymphocystis disease virus (LCDV) to gilthead seabream, Sparus aurata L., larvae was investigated using fertilized eggs from a farm with previous reports of lymphocystis disease. LCDV genome was detected by PCR‐hybridization in blood samples from 17.5% of the asymptomatic gilthead seabream broodstock analysed. Using the same methodology, eggs spawned from these animals were LCDV positive, as well as larvae hatched from them. The presence of infective viral particles was confirmed by cytopathic effects development on SAF‐1 cells. Whole‐mount in situ hybridization (ISH) and immunohistochemistry (IHC) showed the presence of LCDV in the epidermis of larvae hatched from LCDV‐positive eggs. When fertilized eggs were disinfected with iodine, no viral DNA was detected either in eggs (analysed by PCR‐hybridization) or in larvae (PCR‐hybridization and ISH). These results suggest the vertical transmission of LCDV, the virus being transmitted on the egg surface. Larvae hatched from disinfected eggs remain LCDV negative during the endotrophic phase, as showed by PCR‐hybridization, ISH and IHC. After feeding on LCDV‐positive rotifers, viral antigens were observed in the digestive tract, which suggests that viral entry could be achieved via the alimentary canal, and that rotifers can act as a vector in LCDV transmission to gilthead seabream larvae.     

Cloning of a second variant of somatolactin-encoding cDNA from the gilthead seabream Sparus aurata

cDNA clones which include coding sequences of the gilthead seabream (Sparus aurata) somatolactin (SL) have been isolated from a cDNA library prepared from seabream pituitary gland poly (A)⁺ RNA. Flounder – SL cDNA was used as a hybridization probe. The complete nucleotide (nt) sequence of the seabream somatolactin has been determined. The clone encodes a polypeptide of 231 amino acid (aa) residues including 24 amino acid residues of signal peptide. Northern blot hybridization detected one band of approximately 1.8 kb mRNA. By comparing the sequences of this SL cDNA to the one recently published, it is suggested that two variants of the SL exist in seabream. By comparing the sequences of the aa of SL to the deduced aa sequences, it is possible that even a third variant of SL exists in this species.     

Regulation of growth, fatty acid composition and delta 6 desaturase expression by dietary lipids in gilthead seabream larvae (Sparus aurata)

The Δ6 and Δ5 desaturases and elongases show only very limited activity in marine fish, and little is known of the possibility of enhancing Δ6 desaturase gene expression in these fish. The use of plant oils in marine fish diets is limited by their lack of n−3 highly unsaturated fatty acids (HUFA) despite an abundant content of the 18C fatty acid precursor linoleic and α-linolenic acids. The objective of the present study was to determine the ability of larval gilthead seabream to utilize vegetable oils and assess the nutritional regulation of Δ6 desaturase gene expression. Seventeen-day-old gilthead seabream larvae were fed during a 17-day period with one of four different microdiets formulated with either sardine fish oil (FO), soybean, rapeseed or linseed oils, respectively, or a fifth diet containing defatted squid meal and linseed oil. Good larval survival and growth, both in terms of total length and body weight, were obtained by feeding the larvae either rapeseed, soybean or linseed oils. The presence of vegetable oils in the diet increased the levels of 20:2n−9 and 20:2n−6, 18:2n−9, 18:3n−6, 20:3n−6 and 20:4n−6, in larvae fed rapeseed and soybean oils in comparison to those fed FO. In addition, a sixfold increase in the relative expression of Δ6 desaturase-like gene was found in larvae fed rapeseed and soybean oils, denoting the nutritional regulation of desaturase activity through its gene expression in this fish species. However, feeding linseed oil did not increase the expression of the Δ6 desaturase gene to such a high extent.     

Genetic Introgression Between Arctic Charr (Salvelinus Alpinus) and Brook Trout (Salvelinus Fontinalis) in Bavarian Hatchery Stocks Inferred from Nuclear and Mitochondrial DNA Markers

Nuclear insulin-like growth factor 2 gene (IGF-2), growth hormone 1 gene (GH-1) and internal transcribed spacer 1 (ITS-1) of the ribosomal DNA as well as the mitochondrial NADH-3 and NADH-4 dehydrogenase genes (ND-3/4) exhibited species-specific restriction fragment patterns and three microsatellite loci (Sfo18, Ssa85 and Ssa197) had non-overlapping allele size ranges in Arctic charr and brook trout and were used as diagnostic markers for testing genetic purity of hatchery stocks and wild populations of Arctic charr and brook trout in Bavaria, Germany. Screening of four wild populations (three in Arctic charr and one in brook trout) revealed only a single hybrid (back-cross to brook trout) individual in L. Starnberg. In contrast, in three (out of five) hatchery stocks of Arctic charr and in both hatchery stocks of brook trout hybrids were detected with the frequency from 3 to 100%. Three hatchery stocks (SS2, SA and BS1) represent a hybrid swarm because they contained a very high proportion of hybrids (from 83 to 100%) and most or all hybrid individuals had alien alleles at only one or a few of six unlinked diagnostic loci, indicating that post-F₁ hybrids represent the majority of individuals in these stocks and introgression has taken place. Release or escape of introgressed individuals from hatcheries into natural water bodies should be avoided in order to protect the biological diversity and genetic integrity of native fish populations.     

Nauplii Production from Wild,Cultivated, and Mixed Populations of Blue Shrimp,Penaeus stylirostris

ABSTRACT

Due to the low nauplii production of cultivated broodstock, and to reduce the dependence on the wild stock, an experiment was carried out with 400 adult blue shrimp, Penaeus stylirostris, from wild and cultivated (F6) populations. Four treatments, each in duplicate, were applied: (1) wild females and males (W-W); (2) wild females and cultivated males (W-C); (3) cultivated females and wild males (C-W); and (4) cultivated females and males (C-C). More than 300 individual spawns were monitored to evaluate the egg and nauplii production per female. Mixed model ANOVA for factorial arrangements (4 × 3 × 2³ and 3 × 2³) were conducted. The factors considered besides the treatments were: rematuration (number of successive spawnings for a female), ovarian maturity, integrity of the spermatophore attached (complete spermatophore, “wings,” or “remnant”) and condition of spawning (partial or complete). The introduction of both wild females and males was a successful measure to improve the overall egg and nauplii production. Both mixed populations outperformed the cultivated broodstock, but were inferior to the wild stock (average production of eggs and nauplii: W-W-l 12,713 and 34,682, respectively; W-C-l 13,215 and 22,038, respectively; C-W-82,702 and 11,715, respectively; C-C-66,948 and 7,653, respectively). Populations with wild females produce a larger number of eggs, and wild males contribute to higher hatching rates. Other observations indicate the need to select for spawning only those females showing an advanced degree of ovarian maturity, having a complete spermatophore attached to the thelycum, and spawning completely.     

A Novel Zero Discharge Intensive Seawater Recirculating System for the Culture of Marine Fish

Results are presented of a zero‐discharge marine recirculating system used for the culture of gilthead seabream Sparus aurata. Operation of the system without any discharge of water and sludge was enabled by recirculation of effluent water through two separate treatment loops, an aerobic trickling filter and a predominantly anoxic sedimentation basin, followed by a fluidized bed reactor. The fish basin was stocked for the first 6 mo with red tilapia Oreochromis niloticus × O. aureus at an initial density of 16 kg/m³. During this period salinity was raised from 0 to 20 parts per thousand. Then, gilthead seabream, stocked at an initial density of 21 kg/m³, replaced tilapia at day 167 and were cultured for an additional 225 d. Non steady‐state inorganic nitrogen transformations occurred as a result of these salinity changes. After day 210, the system operated at all times with those water quality parameters considered critical for successful operation of mariculture systems, within acceptable limits. Thus ammonia, nitrite, and nitrate concentrations did not exceed 1.0‐mg total ammonia‐N/ L, 0.5‐mg NO₂:‐N/L and 50‐mg NO₃‐N/L, respectively. Sulfide levels in the fish basin were below detection limits and oxygen > 6 mg/L after the oxygen generator was added at day 315. Ammonia, produced in the fish basin and to a lesser extent in the sedimentation basin, was converted to nitrate in the aerobic trickling filter. Nitrate removal took place in the sedimentation basin and to a lesser extent in the fluidized bed reactor. Sludge, remaining in the sedimentation basin at the end of the experimental period, accounted for 9.2% of the total feed dry matter addition to the system. The system was disease‐free for the entire year and fish at harvest were of good quality. Water consumption for production of 1 kg of tilapia was 93 L and 214 L for production of 1 kg of gilthead seabream. Additional growth performance data of gilthead seabream cultured in a similar but larger system are presented. During 164 d of operation of the latter system, maximum stocking densities reached 50 kgl M³ and fish biomass production was 27.7 kg/m³. Relatively poor fish survival and growth resulted from occasional technical failures of this pilot system.