Patterns and selectivity in the feeding of certain mesopelagic fishes

Stomach contents were analysed from the 7 most numerous species of mesopelagic fish caught in a series of 11 hauls over a 24 h period at 230 to 266 m depth in the eastern North Atlantic Ocean. The numerical abundance of organisms per filled stomach and the frequency of occurrence of empty stomachs were used to indicate feeding periodicity. The ecological significance of the feeding periodicity was considered by examining it in connection with an investigation of the day-night vertical distribution of zooplankton and micronekton to 2000 m at the same station. Additional dietary evidence on the 7 species considered was also obtained from the vertical series. Feeding selectivity was examined by comparing the composition of the zooplankton population, sampled separately but simultaneously with the micronekton, with that from the overall stomach contents of the species examined. Feeding periodicity was demonstrated for 6 species, of which 3 were found to be feeding selectively: Valenciennellus tripunctulatus on calanoid copepods, Argyropelecus aculeatus on ostracods, and Lampanyctus cuprarius on amphipods and possibly euphausiids. The limited data available on the other 3 species suggested that they were either random feeders (A. hemigymnus and Lobianchia dofleini) or perhaps selecting against a particular group (Notolychnus valdiviae). No indication of feeding periodicity or selectivity was found for Chauliodus danae. The overall pattern of results confirmed the supposed close correlation between vertical migration and feeding in mesopelagic fish.     

Proximate composition and energy content of mesopelagic fishes from the eastern Gulf of Mexico

The proximate composition of 33 species of mesopelagic fishes collected from the eastern Gulf of Mexico during May–June 1984, July–November 1985 and January 1986 was determined. Water level increased and ash-free dry weight (% dry weight) and protein level (% wet weight) decreased with increasing species' minimum depth of occurrence (that depth below which 90% of a species' population lives). Lipid level (% wet weight or % ash-free dry weight), did not change with species' minimum depth of occurrence. Skeletal ash level (% wet weight) generally decreased with minimum depth of occurrence, whereas carbohydrate level did not change with depth. The variable water level, low lipid level, and high protein level in eastern Gulf of Mexico fishes resulted in a low energy content. These results are generally characteristic of fishes from warm, stable regions of low productivity, such as the eastern North Pacific Gyre. The constant food supply provided by a stable environment may obviate the need for large lipid reserves, in contrast to colder waters at higher latitudes where food availability is seasonal. In addition, the large energy requirements for diel excursions into high-temperature surface waters by the many vertically migrating fishes of this region may influence lipid deposition. The relatively high protein level found in migrators compared to that in non-migrators or weak migrators indicates that locomotory demands influence the percentage of protein found in Gulf fishes. The lack of a positive correlation between protein level and the food availability of a zoogeographic region, suggested in previous studies, is supported here.     

Metabolism,enzymic activities and cold adaptation in Antarctic mesopelagic fishes

Several species of Antarctic mesopelagic fishes that have different minimal depths of occurrence but the same environmental temperature were collected in November–December 1983 and in March 1986 between 0 and 1 000 m in the open water near the marginal ice zone in the vicinity of 60°S 40°W (1983) and 65°S 46°W (1986), and oxygen consumption rate (V _{O 2}) and the activity of two metabolic enzymes, lactate dehydrogenase (LDH, an indicator of the anaerobic potential of locomotory muscle) and citrate synthase (CS, an indicator of citric acid cycle activity or aerobic potential), were determined. In four dominant species, whole-individual oxygen-consumption rate (y, ml O₂ individual^–1 h^–1) varied with weight (X, g) according to the equation y=aX ^b, with b values falling between 0.889 and 1.029. The relation of weight-specific LDH activity (y, U g^–1 wet wt) with weight (x, g) was also described by the equation y=aX ^b, with b values varying between 0.229 and 1.025. Weight-specific CS activity declined with weight, with b values from-0.031 to-0.369. V _{2 O}, LDH activity and CS activity all declined markedly with increased species' minimum depth of occurrence (the depth below which 90% of a species' population lives). Comparisons with previous studies on ecologically equivalent species of the California Borderland indicate that depth-related decreases in metabolism are the result of adapted traits of deeper-living species, not declining temperature within the water column. The metabolic rate of Antarctic mesopelagic fishes is approximately twice that of California species at equivalent temperatures; similar rates were found at the normal habitat temperatures of the two groups. Thus, a well-developed compensation for temperature is present in the Antarctic fishes: cold adaptation. Differences in enzymic activity among species, and among different sized individuals of a species are related to differences in metabolic rate and locomotory capacity. Enzymic indices can be used to estimate metabolic rates and evaluate ecological parameters such as predatory strategies and niche separation.     

Vertical distribution and migration of fishes of the lower mesopelagic zone off Oregon

An exceptionally large midwater trawl (50 m² mouth area) with 5 opening and closing codends was towed horizontally in the lower mesopelagic zone at depths of 500, 650, 800 and 1000 m off Oregon (USA) from 1–6 September, 1978. In comparison to more conventional trawls, ours collected more fish, including rare species and large individuals of common species. Comparison of collections made by day and by night revealed that 12 of the 15 most common species probably migrated vertically. Bathylagus milleri evidently migrates from 650 m during the day to 500 m at night. Cyclothone acclinidens and C. atraria were more abundant by night than by day at 800 m, possibly due to an upward migration from deeper depths at night. C. pseudopallida, C. signata, Chauliodus macouni, Tactostoma macropus and Stenobrachius leucopsarus were more abundant by day than by night at 500 m, suggesting that they migrated out of this depth horizon at night. Lampanyctus regalis, and large individuals of B. pacificus were more abundant by night than by day at 500 m, possibly because they migrated upward from near 650 m. Many species exhibited trends of increasing or decreasing size with depth, and several species showed changes in migratory behavior with size. For example, only small (<240 mm) T. macropus migrated vertically, whereas only large (>110 mm) B. pacificus appeared to migrate. Depths of maximum abundance of congeneric species were usually separated. B. milleri and B. pacificus had similar distributions by day, but the former was shallower at night. S. leucopsarus tended to live shallower than S. nannochir both day and night. Congeners always occurring at the same depth were Cyclothone pseudopallida and C. signata (both most abundant at 500 m) and C. acclinidens and C. atraria (both most abundant at 800 m).     

Protein composition of white skeletal muscle from mesopelagic fishes having different water and protein contents

The consequences for white skeletal muscle of the whole body variation in water and protein content were examined in 11 mesopelagic fishes taken off the coast of Oregon, USA, in 1983. For such muscles, water content varied from 71 to 91% of muscle wet weight, and protein content ranged from 56 to 141 mg g^-1 muscle wet weight, depending on the species. Dilution by increased water content did not account for the decrease in protein content. Total muscle protein was partitioned into soluble (myogen or sarcoplasmic) and insoluble (myofibrillar) components. Both the myogen and myofibrillar components are reduced in muscle with decreased protein content. The activities (units g^-1 wet wt) of white muscle L-lactate dehydrogenase and L-malate dehydrogenase are higher in fishes undergoing diel vertical migration to surface waters than in fishes that either do not migrate or do not migrate to surface waters. The differences in enzyme activities are not due to a general dilution of muscle protein. The actin content of white skeletal muscle was maintained at a relatively constant level in all 11 species examined and was similar to actin levels observed previously in the white skeletal muscle of scombrids and demersal fishes. This conservation of actin content requires species with a reduced muscle protein content to maintain a significant fraction of their total protein as actin. The specific activities of the myofibrillar Mg²⁺–Ca²⁺-activated adenosine triphosphatases of the mesopelagic species are similar in all 11 species studied. Thus, the ratios of proteins in the isolated myofibrils are probably similar. These results suggest that, in species with decreased muscle protein, there is an increase in the non-myofibrillar form of actin.     

Observations on mesopelagic fish from the Red Sea

Mesopelagic fish were collected from a cruise with the R.V. “Dr. Fridtjof Nansen” to the northern Red Sea and the interior Gulf of Aden off the Republic of Djibouti in March 1981. Off the Republic of Djibouti five species of mesopelagic fishes were caught and in the Red Sea six species. Benthosema pterotum (Alcock) were dominant in both areas. In the Red Sea Maurolicus muelleri (Gmelin) were also abundant. The mesopelagic fish caught in the Red Sea showed a pattern of vertical migration similar to that observed in other areas, in spite of the special hydrographical regime. The number of gill rakers of B. pterotum caught in the Red Sea differed significantly from that of all other populations studied. The number of photophores of M. muelleri apparently do not differ much from other populations. B. pterotum from the Red Sea mature at a larger size than other populations, while M. muelleri mature at a smaller size. The relative fecundity of M. muelleri is higher in the Red Sea than in other regions studied, while no significant difference was observed in B. pterotum. Both species appear to be opportunistic feeders. Their feeding chronology in the Red Sea seems to be similar to those observed in other areas.     

Distribution patterns of mesopelagic fishes with special reference to Vinciguerria lucetia Garman 1899 (Phosichthyidae: Pisces) in the Humboldt Current Region off Peru

The horizontal and vertical distributions of adult mesopelagic fishes are described from acoustic and trawl surveys over the full-depth of 500 m at 169 stations on a longitudinal transect crossing the Humboldt Current (03°45′S, 81°76′W and 18°23′S, 71°13′W) at 50–200 nautical miles off Peru during austral spring (October–November) of 2001, 2002 and 2003. A total of 2,952 kg of fishes was collected, which included 13 families, 23 genera and 28 species. The mesopelagic community is dominated by the families Phosichthyidae (Vinciguerria lucetia), Myctophidae (Diogenichthys laternatus and Lampanyctus idostigma) and Bathylagidae (Leuroglossus urotranus), accounting for 60.4, 12.8 and 3.7%, respectively, of the total catch. Based on horizontal distribution patterns these species were categorized into three groups, i.e. northern-central upwelling front group (L. urotranus, Nemichthys fronto and Scopelarchoides nicholsi), Southern upwelling front group (Hygophum reinhardti, Myctophum nitidulum, Paralepis sp and Scopeloberyx sp.) and pan-Humboldt Current group (V. lucetia, D. laternatus, L. (Nannobrachium) idostigma, L. omostigma, M. aurolaternatum, Triphoturus oculeus, Bathylagus (Melanolagus) berycoides, Leuroglossus stilbius, Argyropelecus affinis, Sternoptyx obscura, Melamphaes sp., Stomias sp. and Scopelosaurus sp.). Nighttime vertical distribution was characterized by a single abundance peak in the upper 50 m. Daytime patterns showed three peaks of abundance: an upper peak, in the upper 100 m, a midwater peak between 200 and 400 m, coinciding with an oxygen minimum zone, and a deeper peak between 400 and 500 m. V. lucetia was dominant in the upper and midwater peaks while myctophids, other planctivorous and piscivorous fishes were distributed in the midwater. Acoustic back-scattered energy (S _a) was ubiquitous in the region. Maximum S _a was mainly located between 11° and 18°S during day and night. V. lucetia is a significant component of the sound-scattering layers in the Humboldt Current Region off Peru.     

Comparative studies on the metabolism of shallow-water and deep-sea marine fishes. V. Effects of temperature and hydrostatic pressure on oxygen consumption in the mesopelagic zoarcid Melanostigma pammelas

Measurements have been made of routine oxygen consumption rates ( ) of the mesopelagic deep-sea zoarcid fish Melanostigma pammelas. Determinations were made over ecologically relevant ranges of 3 variables; temperature (3° to 10°C), hydrostatic pressure (1 to 170 atm), and oxygen partial pressure (1 to 160 mm Hg). Weight-specific s were uniformly low. Of the 3 test variables, only temperature had significant metabolic effects within the ranges studied. Q₁₀'s were 6.75 between 3° and 5°C. 1.47 between 5° and 7°C, and 17.4 between 7° and 10°C. These Q₁₀'s were constant over the hydrostatic pressure range studied. Between 3° and 7°C the fish regulated their rates of oxygen consumption down to PO₂'s comparable to those occurring in their natural environments (6 to 12 mm Hg). The showed no capacity to tolerate anoxic conditions. The physiological and ecological significance of these results is discussed, particularly with reference to thermal effects and to the basis of survival of this fish in the oxygen minimum layers of the eastern Pacific Ocean. Since it is possible to maintain M. pammelas in the laboratory for extended periods of time (over 12 months) it could serve as the basis for many interesting studies of deep-sea fish biology.     

Proximate and elemental composition and energy content of mesopelagic crustaceans from the Eastern Gulf of Mexico

The proximate and elemental chemical compositions of 25 species of pelagic decapod and mysid crustaceans collected from the eastern Gulf of Mexico (27°N; 86°W, 1984 to 1989) was examined. Water level ranged from 63 to 95% and increased slightly with species' increased depth of occurrence. Protein levels were generally high (1.5 to 18.3% wet wt, WW; 27.6 to 62.4% ash-free dry wt, AFDW) and comprised the primary organic component in the majority of species. Protein, both as % WW and % AFDW, decreased with increased depth of occurrence. In contrast to protein, lipid levels were low (0.5 to 8.9% WW; 5.7 to 60.9% AFDW), and increased with increased depth of occurrence. Carbon and nitrogen best mirrored measured lipid and protein levels when considered as non-protein carbon and non-chitin nitrogen, respectively. C:N ratios increased with increased depth, consistent with changes in protein and lipid with depth. Because of their compositional attributes, resident Gulf of Mexico species have a low total wet weight energy content relative to species from more productive regions. Energy content showed no significant trend with depth. Vertical migration patterns were distinctly different between shallow-and deep-living gulf species and these differences were largely responsible for the relationships observed between composition and depth. In migrating species, the protein and nitrogen content were higher, the lipid and carbon contents and C:N ratio lower, than in non-migrating species. Three deep-living species of the genus Acanthephyra were found to be compositionally atypical, resembling shallow, migrating types rather than other deep-living, non-migratory species.     

Observations on the nocturnal feeding of some mesopelagic decapod crustacea

Feeding in relation to temporal changes in the depth distribution of predator and prey is described for 9 species of mesopelagic decapods from an examination of 268 foreguts. Intensive nighttime feeding appears to be the rule in all species. The smaller decapods Sergestes (Sergestes) atlanticus, Sergestes (Sergestes) sargassi and Sergestes (Sergestes) pectinatus exploit the smaller prey, principally copepods and to a lesser extent ostracods. Larger decapod species Sergestes (Sergestes) henseni, Sergestes (Sergestes) curvatus, Sergestes (Sergia) grandis, Systellaspis debilis, and Acanthephyra purpurea mainly prey on macrozooplankton and micronekton, i.e., chaetognaths, euphausiids, decapods and fish, but copepods also occur in the foreguts. Gennadas valens is exceptional for the high incidence of foraminiferal remains, and a predator-prey relationship seems probable. All 9 decapod species have mixed diets, and pronounced feeding preferences are not evident. However, a high incidence of “secondary” feeding or “dietary contamination” has been deduced from the frequent occurrence of remains of the copepods Pleuromamma spp. and Oncaea spp. in the foreguts of the larger decapod species. Direct feeding cannot have occurred, since the depth distributions of these copepods and decapods are disjunct by day and night. It is concluded that the remains of Pleuromamma probably represent the food of the larger prey such as chaetognaths etc. which are eaten by the decapods. The presence of Oncaea is speculatively attributed to a possible ectoparasitic relationship with the larger prey items, but confirmatory evidence is required. These anomalies suggest that caution must be exercised in deducing predator-prey relationships simply from gut contents without consideration of distributional factors.     

Distribution and diel vertical movements of mesopelagic scattering layers in the Red Sea

The mesopelagic zone of the Red Sea represents an extreme environment due to low food concentrations, high temperatures and low oxygen waters. Nevertheless, a 38 kHz echosounder identified at least four distinct scattering layers during the daytime, of which the 2 deepest layers resided entirely within the mesopelagic zone. Two of the acoustic layers were found above a mesopelagic oxygen minimum zone (OMZ), one layer overlapped with the OMZ, and one layer was found below the OMZ. Almost all organisms in the deep layers migrated to the near-surface waters during the night. Backscatter from a 300 kHz lowered Acoustic Doppler Current Profiler indicated a layer of zooplankton within the OMZ. They carried out DVM, yet a portion remained at mesopelagic depths during the night. Our acoustic measurements showed that the bulk of the acoustic backscatter was restricted to waters shallower than 800 m, suggesting that most of the biomass in the Red Sea resides above this depth.     

The permeability to oxygen of the swimbladder of the mesopelagic fish Ceratoscopelus maderensis

The permeability to oxygen of the swimbladder of physoclistous fish is important because of the usually high oxygen content of the bladder. The oxygen permeability and guanine content of the swimbladder wall of the mesopelagic myctophid Ceratoscopelus maderensis (Lowe) were measured. The oxygen permeability was 0.0672 cm³ m/atm min cm² and the guanine content was 73 g/cm². Attention is drawn to the high rates of gas loss which would occur from a swimbladder with these characteristics and the problems of maintaining the gas content, especially at depth.     

Spatial variability in lipid composition of calanoid copepods from Fram Strait,the Arctic

The calanoid copepods Calanus hyperboreus and C. finmarchicus were investigated in view of their lipid and wax ester content and their fatty acid and alcohol composition. Analyses were performed in females and copepodid stages V and IV from the Fram Strait region between Greenland and Spitsbergen in 1984. This region offers different food conditions like diatom blooms in the North East Water Polynya, food shortage in areas with very close ice cover, high phytoplankton biomass in the marginal ice zone and lower biomass in the open Atlantic water. Lipids contained generally more than 70% wax esters. Highest levels were found in C. hyperboreus with more than 90%. This percentage was not very variable, in spite of large differences in dry weight and lipid content. Copepods with particularly high weight and lipid content were found in the North East Water Polynya. The lightest individuals were found under the pack ice. Lipid proportions per unit dry weight were higher in C. hyperboreus than in C. finmarchicus, whose lowest values were found in the open Atlantic water. Spatial variability in fatty acid composition was much higher than in alcohol composition. The principle alcohols, 20:1 and 22:1, generally accounting for more than 80% of total alcohols. In the North East Water Polynya, the predominant monounsaturated fatty acid was 16:1, while under the ice 20:1 and 22:1 dominated. In the marginal ice zone and in the open water, the 18:4 acid reached percentages up to 30% of total fatty acids. These changes were related to the different food conditions. C. hyperboreus appears to be best adapted to the cold water and unfavourable conditions of polar regions because of its high lipid and wax ester store with long-chain wax esters of high calorific value.     

Capillary supply and mitochondrial volume density in the axial muscles of the mesopelagic teleost Argyropelecus hemigymnus

Quantitative measurements have been made on the ultra-structure and capillary supply to the axial muscles of the mesopelagic hatchet fish Argyropelecus hemigymnus (Cocco, 1829). Fish were collected at Eastern North Atlantic Ocean Station 10244, 32°48N; 31°15W during November 1980, from a depth of 480 to 550 m. Mitochondria with densely packed cristae occupy 44.3% of slow-fibre volume. Each myofibril is in direct contact with a mitochondrion. Compared with other fishes studied, the capillary supply to A. hemigymnus slow fibres is poorly developed. The average number of capillaries per fibre is 0.9, such that each m of capillary contact supplies 0.011 m² of fibre cross-sectional area. The capillary surface area (m²) supplying 1 m³ of slow-fibre mitochondria is 0.17 in anchovy (Engraulis encrasicolus), 0.14 in rat-fish (Chimaera monstrosa), 0.14 in tench (Tinca tinca), 0.16 in catfish (Clarias mossambica), and only 0.025 for A. hemigymnus. It is suggested that, relative to the former species, some modifications in factors determining tissue oxygenation (e.g. myoglobin concentrations, blood flow, perfusion distribution or haemoglobin) and/or mitochondrial respiration rate are required in order to match oxygen supply and demand to the slow muscle in A. hemigymnus.     

Larval mesopelagic fish assemblages in the Kuroshio–Oyashio transition region of the western North Pacific

We examined larval mesopelagic fish assemblages, their distribution, and seasonal occurrence patterns in the Kuroshio–Oyashio transition region of the western North Pacific where complex hydrographic structures are observed due to the confluence of the Kuroshio Extension and Oyashio current. Larvae of the dominant families Myctophidae, Gonostomatidae, Bathylagidae, Sternoptychidae, and Phosichthyidae were represented by 31 species or types belonging to 24 genera. Based on species composition analysis using the Morishita–Horn similarity index, five assemblages were recognized: Oyashio, Spring Transition, Summer Transition, Kuroshio, and Slope Water assemblages. The distribution patterns of these assemblages corresponded closely with hydrographic structures such as position of the Oyashio and Kuroshio Extension fronts, warm core rings and streamers. Spring Transition (April) and Summer Transition (July–October) assemblages were the most important larval assemblages in the transition region. Larval abundances were low during late autumn and winter. The Spring Transition and Summer Transition assemblages were composed of subtropical, transitional, subarctic, and slope-water species, suggesting the importance of the transition region as nursery grounds of mesopelagic fishes of various origins from subarctic to subtropical waters. Larval fish transport by the Kuroshio, Oyashio, and Tsugaru Warm currents into the transition region is discussed.     

Persistent near-bottom aggregations of mesopelagic animals along the North Carolina and Virginia continental slopes

Submersible observations during four missions over the North Carolina and Virginia continental slopes (184–900 m) documented the occurrence of large aggregations of mesopelagic fishes and macronektonic invertebrates near or on the bottom. Aggregated mesopelagics formed a layer up to tens of meters deep positioned from a few centimeters to 20 m, usually <10 m, above the substrate. Aggregations were numerically dominated by microvores, notably the myctophid fish Ceratoscopelus maderensis and the penaeid shrimp Sergestes arcticus. Consistently present but in relatively lower numbers, were mesopelagic predators, including the paralepidids Notolepis rissoi and Lestidium atlanticum, the eel Nemichthys scolopaceus, the stomiid fishes Chauliodus sloani and Stomias boa ferox, and squids Illex spp. Near-bottom aggregations do not appear to be an artifact due to attraction to the submersible. Based on submersible observations in three areas in 4 years spanning a decade, near-bottom aggregations of midwater organisms appear to be a geographically widespread and persistent phenomenon along the continental slope of the southeastern US Aggregations may exploit areas of enhanced food resources at the bottom.     

Early development and estimated life cycle of the mesopelagic copepod Pareuchaeta elongata in the southern Japan Sea

Life cycle of the mesopelagic copepod Pareuchaeta elongata was investigated combining laboratory rearing data on the eggs, nauplii, and early copepodites with field growth data on the late copepodites. Examination and incubation at near the in situ temperature (0.5°C) of egg sacs collected from the field indicated that the clutch size was 13 to 24 eggs (mean: 20), hatching time was 39.4d (mean), and hatchability was low (mean: 28.5%). The development time at 0.5°C was 2.4 d for Nauplius Stage 1 (N1), 4.6 d for N2, 6.2 d for N3. 7.4 d for N4, 7.1 d for N5, 20.8 d for N6, 36.7 d for Copepodite Stage 1 (C1) and 65.3 d for C2. From the numerical analysis of seasonal samples collected from the field, the development time was estimated as 1 mo for C3, 2 mo for C4 and 2.5 mo for C5. Thus, the egg hatching time plus the integrated development time by stage was 355 d or nearly 1 yr (i.e. span of one life cycle). Duration of the C6 (adult) was estimated as>2.5 mo. Combining the present data on development times of each stage with published data on the major spawning season (August to October) and ontogenetic migration, a schematic representation of the life cycle of this copepod was developed. Between-stage comparison of wet, dry, and ash-free dry weights of all developmental stages of preserved wild specimens revealed that there is almost no gain in weight during naupliar stages, and the greatest weight increment over the life cycle was during the C4 stage. The present results are compared with those for the same and related species living in other regions.     

Age and growth of the mesopelagic squid Ancistrocheirus lesueurii (Oegopsida: Ancistrocheiridae) from the central-east Atlantic based on statolith microstructure

Statolith microstructure was studied in 56 Ancistrocheirus lesueurii (25 to 423 mm of mantle length, ML) caught in the central-east Atlantic. Statolith growth increments were grouped into three main growth zones, distinguished mainly by increment width. The second transition in the statolith microstructure (from Zone 2 to Zone 3) coincides with the life history shift from epipelagic and upper mesopelagic to a bathyal habitat. Second-order bands (mean 27.65 growth increments) and sub-bands (mean 13.6 growth increments) within statolith microstructure appeared to be related to the lunar cycle. Striking sexual dimorphism is reflected in the age and growth rates: males live ca. 1 yr, while females only start maturing at this age and obviously live >1.5 yr. A. lesueurii is a slow growing squid, attaining 25 to 30 mm ML at the age of 100 d. After ontogenetic migrations into bathypelagic waters at ML > 30 to 35 mm, growth rates gradually decrease to the minimum known values for squids. Based on back-calculated hatching dates, A. lesueurii hatches throughout the year with a peak between November and March. Received: 28 August 1996 / Accepted: 31 January 1997