Dynamic changes in DNA methylation during seahorse (Hippocampus reidi) postnatal development and settlement.

INTRODUCTION: Most living marine organisms have a biphasic life cycle dependent on metamorphosis and settlement. These critical life-history events mean that a developmentally competent larva undergoes a range of coordinated morphological and physiological changes that are in synchrony with the ecological transition from a pelagic to a benthonic lifestyle. Therefore, transition from a pelagic to a benthonic habitat requires multiple adaptations, however, the underlying mechanisms regulating this process still remains unclear. Epigenetic regulation and specifically DNA methylation, has been suggested to be particularly important for organisms to adapt to new environments. Seahorses (Family Syngnathidae, Genus Hippocampus) are a fascinating group of fish, distinguished by their unique anatomical features, reproductive strategy and behavior. They are unique among vertebrate species due to their "male pregnancy", where males nourish developing embryos and larvae in a brood pouch until hatching and parturition occurs. After birth, free-swimming offspring are pelagic and subsequently they change into a demersal lifestyle. Therefore, to begin to address the question whether epigenetic processes could be involved in the transition from a planktonic to a benthonic lifestyle observed in seahorses, we studied global DNA methylation profiles in a tropical seahorse species (Hippocampus reidi) during postnatal development and settlement. RESULTS: We performed methylation-sensitive amplified polymorphism (MSAP) along with quantitative expression analysis for genes suggested to be involved in the methylation machinery at six age groups: 1, 5, 10, 20, 30 and 40 days after male's pouch release (DAR). Results revealed that the H. reidi genome has a significantly different DNA methylation profile during postnatal development and settlement on demersal habitats. Moreover, gene expression analysis showed up- and down-regulation of specific DNA methyltransferases (DNMTs) encoding genes. CONCLUSION: Our data show that the differences in the DNA methylation patterns seen among developmental stages and during the transition from a pelagic to a benthonic lifestyle suggest a potential for epigenetic regulation of gene expression (through DNA methylation) in this species. Therefore, epigenetic mechanisms could be necessary for seahorse settlement. Nevertheless, if these epigenetic mechanisms come from internal or if they are initiated via external environmental cues should be further investigated.

Does acidification lead to impairments on oxidative status and survival of orange clownfish Amphiprion percula juveniles?

The nitrification process in recirculating aquaculture systems can reduce water pH. Fish can also be exposed to water acidification during transport, an important feature in the aquarium industry, as live fish can be kept in a closed environment for more than 24 h during overseas aerial transportation. Therefore, it is important to study the responses of fish to acidic environments. We investigated the impacts of acute exposure to decreasing pH levels in orange clownfish Amphiprion percula juveniles on their survival and oxidative stress status. Fish were exposed to pH 5, 6, 7, and 8 for 96 h. We observed a significant reduction in survival (85%) and protein damage as measured by P-SH (protein thiol) for fish maintained at pH 5. Despite no effects on survival or oxidative damage, fish exposed to pH 6 showed an increase in their antioxidant defense systems, demonstrating this pH level could not be suitable for them as well. Furthermore, there were no negative effects for fish kept at pH 7, compared to those maintained at pH 8 during this short-term evaluation.

Histological development of the long-snouted seahorse Hippocampus guttulatus during ontogeny.

The objective of the present study was to describe histological development of the European long-snouted seahorse Hippocampus guttulatus, to increase understanding of the biology and physiology of the species. Most vital organs were present in juveniles by the time of their release from the male's pouch. Digestive tract specialization occurred at 89 effective day-degrees (D°eff ), corresponding to 15 days post partum (dpp), with development of the first intestinal loop and mucosal folding. At 118 D°eff (20 dpp), lipids were being mobilized from the liver and oocytes attained the perinuclear stage. The fovea emerged at 177 D°eff (30 dpp), contemporaneous with the shift from pelagic to benthic behaviour in juveniles. At this stage, the most interesting feature was the formation of the second intestinal loop. Male gonads were never observed during the study (from 0 to 354 D°eff ; 0-60 dpp), but the first oogonia were present at 30 D°eff (5 dpp). In 354 D°eff (60 dpp) juveniles, oocytes were observed in a cortical alveoli stage, indicating maturity. Low digestive efficiency was observed at early stages, which was due to a poorly developed gastrointestinal tract and an immature digestive tract prior to 89 D°eff . The present study demonstrates that approximately 89 and 177 D°eff represent two important transitional stages in the early development of H. guttulatus. At a temperature of approximately 19 ± 1°C and an age of 1 month (177 D°eff ), main organs were fully functional, suggesting that the adult phenotype was largely established by that age, with females becoming mature at the age of 2 months (354 D°eff ).

Mycobacterium hippocampi sp. nov., a rapidly growing scotochromogenic species isolated from a seahorse with tail rot.

A Gram-positive, aerobic, non-motile, non-sporulating, acid-fast, and rod-shaped bacterium (BFLP-6(T)), previously isolated from a seahorse (Hippocampus guttulatus) with tail rot, was studied using a polyphasic taxonomic approach. Growth occurred at 15-35 °C (optimum 25 °C), at pH 5.0-10.0 (optimum pH 7.0) and at NaCl concentrations between 0 and 6 % (w/v). The G+C content of DNA was 66.7 mol%. The predominant fatty acids were C(18:1) ω9c, C(16:0) and C(16:1) ω6c. A mycolic acid pattern of alpha-mycolates and keto-mycolates was detected. Analysis of concatenated sequences (16S rRNA, rpoB, ssrA and tuf genes), and chemotaxonomic and phenotypic features indicated that strain BFLP-6(T) represents a novel species within the genus Mycobacterium, for which the name Mycobacterium hippocampi sp. nov. is proposed. The type strain is BFLP-6(T) (=DSM 45391(T) =LMG 25372(T)).

Occurrence of Seahorses Hippocampus spp. in the Southernmost Part of Western Europe: A New Maximum Depth Record.

Achieving sustainable resource use is a priority to meet future challenges. The Gulf of Cádiz, located in the Atlantic waters of southern Europe, is home to a significant fishing fleet due to the richness and diversity of its ecosystems. Managing this area is complex due to the diversity of variables, including social, ecological, and oceanographic factors. Therefore, multidisciplinary approaches are proposed for implementing conservation strategies. One strategy for defining area-based management measures is through the use of flagship species, such as seahorses. These emblematic animals can assist in defining such measures. However, there is currently scarce information on the occurrence of seahorses in the Gulf of Cádiz. In this study, we present the first occurrence data of two species of the genus Hippocampus (H. hippocampus and H. guttulatus) in this area. The Gulf of Cádiz is not only described as the southernmost region of their distribution in continental Europe, but it is also a significant landmark for the genus Hippocampus, as one adult was captured at a depth of up to 101 m. Five management areas based on differentiated benthic habitats are proposed. We believe that our study has the potential to significantly improve conservation of seahorses and induce a positive impact on the ecosystem.

Oceanibacterium hippocampi gen. nov., sp. nov., isolated from cutaneous mucus of wild seahorses (Hippocampus guttulatus).

A Gram-negative, aerobic, motile and slightly curved rod-shaped bacterium (BFLP-8(T)) was isolated from cutaneous mucus of wild long-snouted seahorses (Hippocampus guttulatus) captured in northwest Spain (Toralla, Galicia). Strain BFLP-8(T) grew at 10-35 °C and pH 5-9 (optimally at 25 °C and pH 7.0) and with 1-6 % (w/v) NaCl (optimally with 2 % NaCl). The predominant respiratory quinone (90 %) was ubiquinone with ten isoprene units (Q-10) and the major fatty acids identified were C(18:1) ω7c (54.8 % of the total), C(19:0) cyclo ω8c (11.6 %), C(16:0) (9.5 %), C(18:1) 2-OH (7.1 %) and C(16:1) ω11c (6.7 %). The G+C content of the DNA was 57.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain BFLP-8(T) formed a distinct clade within the family Sneathiellaceae but is not specifically associated with any species in the family. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain BFLP-8(T) represents a novel species within a new genus, for which the name Oceanibacterium hippocampi gen. nov., sp. nov. is proposed. The type strain is BFLP-8(T) (=CECT 7691(T) = DSM 23444(T)).

Ecological Traits and Trophic Plasticity in The Greater Pipefish Syngnathus acus in the NW Iberian Peninsula.

The great pipefish Syngnathus acus is one of the most representative European syngnathids, being highly associated with seagrass and macroalgal beds. Surprisingly, the ecology of this large ovoviviparous marine fish has received scanty attention. The population inhabiting three sites on Cíes Archipelago (Atlantic Islands National Park, NW Spain) was monitored in 2017-2018 for spatial and temporal changes in abundances, reproduction traits, trophic niche occupancy, and dietary regimes across reproduction states, through an isotopic (δ13C and δ15N) approach. Abundances were highly variable across seasons and sites, decreasing significantly from mid-autumn. The population consisted almost exclusively of large adults that migrate by the end of the breeding season, which extended from mid-spring to summer. Operational sex ratios suggest that the species is sex-role reversed. S. acus is a secondary consumer (Trophic position = 3.36 ± 0.05), preferring amphipods but displaying annual and seasonal dietary plasticity. Mature fish were less selective than immatures (especially females), with a higher preference for amphipods (36-68%) in the former. The second most-preferred prey were carideans, copepods, or isopods, depending on the year and the reproduction state. Overall, the wider trophic niches in females and immature specimens compared to males and mature fish would indicate a higher variability in both the use of prey resources and/or their origin. The present study highlights the trophic plasticity and unique features of S. acus population in the Cíes Archipelago, especially regarding the outstanding size of the fish and the exceptional occurrence of breeders.

Sustainable Aquaculture: Nutrition Studies in Early Developing Finfish, Ornamentals and Experimental Model Fish.

Enhancing the knowledge of feeding/nutritional requirements is key in the growth of fish development and for the optimization of rearing techniques [...].

Turnover Rates and Diet-Tissue Discrimination Factors of Nitrogen and Carbon Stable Isotopes in Seahorse Hippocampus reidi Juveniles Following a Laboratory Diet Shift.

The initial development of seahorse juveniles is characterized by low digestion capabilities. Stable isotope analysis is an effective tool in studies of trophic food webs and animal feeding patterns. The present study provides new insights for the understanding of growth and food assimilation in early developing seahorses following a laboratory diet switch. The study was performed in the early life stages of the seahorse Hippocampus reidi by assessing the influence of diet shift on changes and turnovers in carbon (δ13C) and nitrogen (δ15N) stable isotope in juveniles. Newborn seahorses were fed for 60 days following two feeding schedules (A6 and A11) based initially on copepods Acartia tonsa and subsequently on Artemia nauplii (since days 6 and 11, respectively). After the prey shift, we determined δ13C and δ15N turnover rates as functions of change in either body mass (fitting model G) and days of development (fitting model D), contributions of metabolism and growth to those turnover rates, and diet-tissue discrimination factors. Survival, final dry weight, and final standard length for diet A11 were higher compared to diet A6. The shift from copepods to Artemia led to fast initial enrichments in δ13C and δ15N. Afterwards, the enrichment was gradually reduced until the isotopic equilibrium with the diet was reached. In most cases, both fitting models performed similarly. The isotopic analysis revealed that 100% of tissue turnover was attributed to growth in diet A11, whereas 19-25% was linked to metabolism in diet A6. Diet-tissue discrimination factors were estimated for the first time in seahorse juveniles, resulting in higher estimates for diet A11 (2.9 ± 0.7‱ for δ13C; 2.5 ± 0.2‱ for δ15N) than in diet A6 (1.8 ± 0.1‱ for δ13C; 1.9 ± 0.1‱ for δ15N). This study highlights the relevance of feeding on copepods and their effect on isotopic patterns and discrimination factors in seahorse juveniles after a dietary shift. Regarding the application of the results achieved in relation to the feeding schedules in the rearing of H. reidi, a long period of feeding on copepods during the first days of development is highly recommended.

Implications of Salinity and Acidic Environments on Fitness and Oxidative Stress Parameters in Early Developing Seahorses Hippocampus reidi.

Water acidification affects aquatic species, both in natural environmental conditions and in ex situ rearing production systems. The chronic effects of acidic conditions (pH 6.5 vs. pH 8.0) in seahorses (Hippocampus spp.) are not well known, especially when coupled with salinity interaction. This study investigated the implications of pH on the growth and oxidative stress in the seahorse Hippocampus reidi (Ginsburg, 1933), one of the most important seahorse species in the ornamental trade. Two trials were carried out in juveniles (0-21 and 21-50 DAR-days after the male's pouch release) reared under acid (6.5) and control (8.0) pH, both in brackish water (BW-salinity 11) and seawater (SW-salinity 33). In the first trial (0-21 DAR), there was no effect of pH on the growth of seahorses reared in SW, but the survival rate was higher for juveniles raised in SW at pH 6.5. However, the growth and survival of juveniles reared in BW were impaired at pH 6.5. Compared to SW conditions, the levels of superoxide dismutase and DT-diaphorase, as well as the oxidative stress index, increased for juveniles reared in BW. In the second trial, seahorse juveniles were reared in SW at pH 8.0, and subsequently kept for four weeks (from 21 to 50 DAR) at pH 6.5 and 8.0. The final survival rates and condition index were similar in both treatments. However, the growth under acidic conditions was higher than at pH 8.0. In conclusion, this study highlights that survival, growth, and oxidative status condition was enhanced in seahorse juveniles reared in SW under acidic conditions (pH = 6.5). The concurrent conditions of acidic pH (6.5) and BW should be avoided due to harmful effects on the fitness and development of seahorse juveniles.

First evidence of ingestion and retention of microplastics in seahorses (Hippocampus reidi) using copepods (Acartia tonsa) as transfer vectors.

Microplastics (MPs) are a major concern for marine ecosystems since they can be ingested by a wide range of marine species and transmitted through the food web. However, the potential hazardous impact of MPs in fishes, especially in early developing stages, is relatively unknown. In the present study, we assessed for the first time the ingestion and retention of MPs in early developing seahorses Hippocampus reidi. Seahorses are vulnerable species that may also be affected by both the direct ingestion of MPs through their preys and the accidental ingestion of MPs particles present in the water (i.e., seahorses ingest the prey by suction). We used copepods as both preys for seahorse juveniles and transfer vectors of MPs. Fed or starved copepods previously exposed to polyethylene microspheres (1-5 µm in diameter; 10 and 100 µg L-1) for 60 min at 26 °C showed fast evacuation of microspheres. The presence of MPs in copepods was significantly higher in previously fasted copepods compared to fed copepods. Seahorse juveniles fed on copepods pre-exposed to MPs, accumulated MPs in the gut proportionally to the concentration of MPs in copepods. A lower concentration of MPs in seahorses was observed at the longer exposure time (60 min), especially in fish fed with fasted copepods. However, after longer exposure, MPs were mainly accumulated close to the anus both individually or forming aggregates. Further studies should be performed to assess secondary effects of MPs ingestion in seahorses since they are considered a flagship species for marine conservation.

Nutrient Incorporation in First Feeding Seahorses Evidenced by Stable Carbon Isotopes.

Nutritional issues are among the most critical factors in the initial survival of juvenile seahorses. Currently, there is a knowledge gap on the relationship between nutrient assimilation and the effects on initial mortalities and growth. In the present study, the stable isotope approach was used to assess the incorporation of two live preys (Artemia and copepods) in juvenile seahorses Hippocampus guttulatus. The changes in stable carbon isotope (δ13C) values were studied through two feeding experiments: feeding on Artemia or copepods (experiment 1), and shifting feeding from copepods to Artemia (experiment 2). In experiment 1, after 24-48 h of feeding, juvenile seahorses exhibited small but progressive changes in δ13C values towards those of the corresponding diet, indicating that the assimilation of the food offered was progressively enhanced from days 2-3. Similarly, in experiment 2, a diet shifting from copepods to Artemia caused an increase in δ13C values, reflecting a switch towards the isotopically enriched new diet (Artemia metanauplii). Differences in the assimilation efficiency of preys offered are discussed based on growth and survival rates. The enhanced growth performances and survivals achieved when the juveniles were fed on copepods could be related to higher efficient assimilation of copepods compared to Artemia. The present study demonstrates that the consumption and further assimilation of preys by juvenile seahorses could be traced using stable carbon isotopes. The research on nutrient assimilation of juvenile seahorses should enhance our knowledge on nutrient processes in developing seahorses for a better understanding of initial ontogeny in the early life stages of the species.

Successful Use of Geochemical Tools to Trace the Geographic Origin of Long-Snouted Seahorse Hippocampus guttulatus Raised in Captivity.

The global market of dried seahorses mainly supplies Traditional Chinese Medicine and still relies on blurry trade chains that often cover less sustainable practices targeting these pricey and endangered fish. As such, reliable tools that allow the enforcement of traceability, namely to confirm the geographic origin of traded seahorses, are urgently needed. The present study evaluated the use of elemental fingerprints (EF) in the bony structures of long-snouted seahorses Hippocampus guttulatus raised in captivity in two different locations (southern Portugal and Northern Spain) to discriminate their geographic origin. The EF of different body parts of H. guttulatus were also evaluated as potential proxies for the EF of the whole body, in order to allow the analysis of damaged specimens and avoid the use of whole specimens for analysis. The contrasting EF of H. guttulatus raised in the two locations allowed their reliable discrimination. Although no single body part exactly mimicked the EF of the whole body, seahorse trunks, as well as damaged specimens, could still be correctly allocated to their geographic origin. This promising forensic approach to discriminate the geographic origin of seahorses raised in captivity should now be validated for wild conspecifics originating from different locations, as well as for other species within genus Hippocampus.

A multidisciplinary approach to identify priority areas for the monitoring of a vulnerable family of fishes in Spanish Marine National Parks.

BACKGROUND: Syngnathid fishes (Actinopterygii, Syngnathidae) are flagship species strongly associated with seaweed and seagrass habitats. Seahorses and pipefishes are highly vulnerable to anthropogenic and environmental disturbances, but most species are currently Data Deficient according to the IUCN (2019), requiring more biological and ecological research. This study provides the first insights into syngnathid populations in the two marine Spanish National Parks (PNIA-Atlantic- and PNAC-Mediterranean). Fishes were collected periodically, marked, morphologically identified, analysed for size, weight, sex and sexual maturity, and sampled for stable isotope and genetic identification. Due the scarcity of previous information, habitat characteristics were also assessed in PNIA. RESULTS: Syngnathid diversity and abundance were low, with two species identified in PNIA (Hippocampus guttulatus and Syngnathus acus) and four in PNAC (S. abaster, S. acus, S. typhle and Nerophis maculatus). Syngnathids from both National Parks (NP) differed isotopically, with much lower δ15N in PNAC than in PNIA. The dominant species were S. abaster in PNAC and S. acus in PNIA. Syngnathids preferred less exposed sites in macroalgal assemblages in PNIA and Cymodocea meadows in PNAC. The occurrence of very large specimens, the absence of small-medium sizes and the isotopic comparison with a nearby population suggest that the population of Syngnathus acus (the dominant syngnathid in PNIA) mainly comprised breeders that migrate seasonally. Mitochondrial cytochrome b sequence variants were detected for H. guttulatus, S. acus, and S. abaster, and a novel 16S rDNA haplotype was obtained in N. maculatus. Our data suggest the presence of a cryptic divergent mitochondrial lineage of Syngnathus abaster species in PNAC. CONCLUSIONS: This is the first multidisciplinary approach to the study of syngnathids in Spanish marine NPs. Habitat preferences and population characteristics in both NPs differed. Further studies are needed to assess the occurrence of a species complex for S. abaster, discarding potential misidentifications of genus Syngnathus in PNAC, and evaluate migratory events in PNIA. We propose several preferential sites in both NPs for future monitoring of syngnathid populations and some recommendations for their conservation.

Isolation of Vibrio alginolyticus and Vibrio splendidus from captive-bred seahorses with disease symptoms.

Vibrio species isolated from diseased seahorses were characterized by PCR amplification of repetitive bacterial DNA elements (rep-PCR) and identified by 16S ribosomal RNA gene sequence analysis. The results demonstrated that Vibrio alginolyticus and Vibrio splendidus were predominant in the lesions of these seahorses. To our knowledge, this is the first time that these bacterial species have been associated with disease symptoms in captive-bred seahorses.

Application of Effective Day Degrees in the Assessment of Stable Isotope Patterns in Developing Seahorses under Different Temperatures.

Relations between nutrient assimilation and growth rate in fishes may vary with abiotic factors such as temperature. The effects of feeding status, ontogeny and temperature regimes (15, 18 and 21 °C) on stable isotope (δ13C and δ15N) patterns were assessed in juveniles of the seahorse Hippocampus guttulatus. The use of effective day degrees (D°eff), day degrees (D°) and chronological time (age) were compared as development progress indices. Newborn seahorses were maintained at three temperature levels both deprived of food (5 days) or fed (30 days) on copepods or/and Artemia. Isotopic signatures in fed seahorses clearly differed from those in unfed juveniles. Temperature had a significant effect on δ13C values in fed juveniles throughout the experimental period. δ15N values also varied significantly with age, but not with temperature level. Faster growth and food assimilation in seahorses held at 18 and 21 °C were supported by faster variations in isotopic values. Our findings demonstrate that effective day degrees should be preferred over chronological time as index of developmental progress in temperature fluctuating scenarios or for comparative studios.

Effects of Tissue Preservation on Carbon and Nitrogen Stable Isotope Signatures in Syngnathid Fishes and Prey.

Isotopic stable analysis (SIA) is a powerful tool in the assessment of different types of ecological and physiological studies. For that, different preservation methods for sampled materials are commonly used prior to isotopic analysis. The effects of various preservation methods (freezing, ethanol and formaldehyde) were analyzed for C:N, and δ13C and δ15N signals on a variety of tissues including dorsal fins (three seahorse and two pipefish species), seahorse newborns (three seahorses species), and prey (copepods and different stages of Artemia) commonly used to feed the fishes under rearing conditions. The aims of the study were: (i) to evaluate isotopic effects of chemical preservation methods across different types of organisms and tissues, using frozen samples as controls, and (ii) to construct the first conversion models available in syngnathid fishes. The chemical preservation in ethanol and, to a lesser extent, in formaldehyde significantly affected δ13C values, whereas the effects on δ15N signatures were negligible. Due to their low lipid content, the isotopic signals in fish fins was almost unaffected, supporting the suitability of dorsal fins as the most convenient material in isotopic studies on vulnerable species such as syngnathids. The regression equations provided resulted convenient for the successful conversion of δ13C between preservation treatments. Our results indicate that the normalization of δ15N signatures in preserved samples is unnecessary. The conversion models should be applicable in isotopic field studies, laboratory experiments, and specimens of historical collections.

Conservation genetics of threatened Hippocampus guttulatus in vulnerable habitats in NW Spain: temporal and spatial stability of wild populations with flexible polygamous mating system in captivity.

This study was focused on conservation genetics of threatened Hippocampus guttulatus on the Atlantic coast of NW Iberian Peninsula. Information about spatial structure and temporal stability of wild populations was obtained based on microsatellite markers, and used for monitoring a captive breeding program firstly initiated in this zone at the facilities of the Institute of Marine Research (Vigo, Spain). No significant major genetic structure was observed regarding the biogeographical barrier of Cape Finisterre. However, two management units under continuous gene flow are proposed based on the allelic differentiation between South-Atlantic and Cantabrian subpopulations, with small to moderate contemporary effective size based on single-sample methods. Temporal stability was observed in South-Atlantic population samples of H. guttulatus for the six-year period studied, suggesting large enough effective population size to buffer the effects of genetic drift within the time frame of three generations. Genetic analysis of wild breeders and offspring in captivity since 2009 allowed us to monitor the breeding program founded in 2006 in NW Spain for this species. Similar genetic diversity in the renewed and founder broodstock, regarding the wild population of origin, supports suitable renewal and rearing processes to maintain genetic variation in captivity. Genetic parentage proved single-brood monogamy in the wild and in captivity, but flexible short- and long-term mating system under captive conditions, from strict monogamy to polygamy within and/or among breeding seasons. Family analysis showed high reproductive success in captivity under genetic management assisted by molecular relatedness estimates to avoid inbreeding. This study provides genetic information about H. guttulatus in the wild and captivity within an uncovered geographical range for this data deficient species, to be taken into account for management and conservation purposes.

Use of multivariate analysis to assess the nutritional condition of fish larvae from nucleic acids and protein content.

The nutritional condition of turbot larvae (Scophthalmus maximus) was assessed by a multivariate analysis with DNA, RNA, and protein content as input variables. Special attention was given to the time that feeding began and to the timing and duration of starvation. The combination of the principal components analysis and the stepwise discriminant analysis, both techniques of multivariate analysis, made it possible to allocate the larvae into groups that were defined and identified based on similarities in developmental stage and nutritional condition. The developmental stage was mostly determined by the input variables DNA and protein content, while nutritional condition was determined by the RNA content. In the period studied, the more developed larvae were less resistant to starvation. Furthermore, when initial feeding was delayed as little as 6 h, the variables analyzed were markedly changed, and the effect on the deprived larvae was found to be equivalent to a 3-day delay in development-when compared with the larvae fed immediately after mouth opening. Through this technique, new samples of larvae with unknown history might be classified into groups, using their DNA, RNA, and protein content as input values in the defined classification functions. Results were compared to those obtained using RNA/DNA and RNA/dry weight indices, and the multivariate method was considered to be more sensitive and to provide extra information about larval nutritional history and development.