Profile of the gut microbiota of Pacific white shrimp under industrial indoor farming system.

The gut microbial communities interact with the host immunity and physiological functions. In this study, we investigated the bacterial composition in Litopenaeus vannamei shrimp's gut and rearing water under different host (developmental stage: juvenile and adult; health status: healthy and diseased) and environmental factors (temperature 25 °C and 28 °C; and light intensity: low and high). The PCoA analysis showed that all water samples were clustered together in a quarter, whereas the gut samples spread among three quarters. In terms of functional bacteria, gut samples of adult shrimp, healthy adult shrimp, adult shrimp raised at 28 °C, and juvenile shrimp under high light intensity exhibited a higher abundance of Vibrionaceae compared to each other opposite group. Gut samples of juvenile shrimp, infected adult shrimp, juvenile shrimp with low light intensity, and adult shrimp with a water temperature of 25 °C showed a higher abundance of Pseudoaltromonadaceae bacteria compared to each other opposite group. Gut samples of juvenile shrimp, healthy adult shrimp, adult shrimp raised at a water temperature of 28 °C, and juvenile shrimp with high light intensity showed the higher abundance of Firmicutes/Bacteroidota ratio compared to each other opposite group. Our results showed that L. vannamei juveniles are more sensitive to bacterial infections; besides, water temperature of 28 °C and high light intensity groups were both important conditions improving the shrimp gut bacterial composition under industrial indoor farming systems. KEY POINTS: • Bacteria diversity was higher among shrimp intestinal microbiota compared to the rearing water. • Shrimp juveniles are more sensitive to bacterial infection compared to adults. • Water temperature of 28 °C and high light intensity are recommended conditions for white shrimp aquaculture.

Long-Term Exposure of the Red Cherry Shrimp Neocaridina davidi to Diclofenac: Impact on Survival, Growth, and Reproductive Potential.

The current study was aimed at studying the long-term effects of diclofenac on the freshwater shrimp Neocaridina davidi, concerning survival, somatic growth, and reproduction. In this study, both ovigerous females and males of this species were exposed for 63 d to 0 (control), 0.1, or 1 mg/L of diclofenac. At the highest concentration, significant mortality was detected, and the somatic growth of females was significantly decreased. The percentage of females with a second spawn, observable from day 45, significantly increased at 1 mg/L, while the time between spawns was significantly reduced at both concentrations assayed. However, the gonadal analysis made at the end of the assay in the surviving females showed a significantly lower proportion of advanced oocytes in females exposed to 1 mg/L, as compared to control. Concerning hatching, the percentage of ovigerous females that could have successful hatching was reduced at 1 mg/L of diclofenac, especially for the first spawn. For the second spawn, the low number of juveniles hatched from females exposed to 1 mg/L also showed a significantly higher incidence of morphological abnormalities, such as hydropsy and underdeveloped appendages. Taken together, these results showed that even when diclofenac was able to produce earlier spawns, the reproductive output of each spawn was reduced.

Brothers are better than nothing: first report of incestuous mating and inbreeding depression in a freshwater decapod crustacean.

This study aimed at evaluating the effect of one generation of full-sibling mating on traits related to the fitness of the gregarious freshwater shrimp Neocaridina davidi, both under optimal and stressful (i.e. starvation) laboratory conditions. Females were maintained either with their brothers (Inbreeding treatment) or non-brothers (Outbreeding treatment), and the first and second broods were used to evaluate egg production and juvenile quality, respectively. The latter was analyzed in a 60-day period following hatching under optimal rearing conditions, and in a 20-day period following hatching under food deprivation conditions. All surviving females from both treatments mated and spawned, indicating that mating with brothers was as likely as mating with non-brothers. With respect to offspring production, inbreeding had no effect on fecundity and fertilization success, but negatively affected the number of hatched juveniles. These results suggest that egg loss was higher in inbred clutches, possibly due to lower embryonic survival. On the other hand, the effect of inbreeding on growth was absent for embryos and for juveniles under optimal rearing conditions, while it was significant for juveniles starved for 10 consecutive days following hatching. Inbreeding depression for survival was only detected in juveniles from stages S8-S9 under optimal rearing conditions. Overall, the present results show that N. davidi fitness decreases after one generation of full-sibling mating. The use of a potentially weak food deprivation protocol and/or the evaluation of only one inbred generation could explain the absence or subtle inbreeding depression for some of the evaluated traits. No evident relationship between life-history traits and the existence and magnitude of inbreeding was found when comparing our results with those previously reported in high-fecundity marine crustaceans and low-fecundity terrestrial crustaceans.

Female energy dynamics in the southernmost fiddler crab: Mixed breeding strategy in Leptuca uruguayensis.

At the south of its geographical distribution, the fiddler crab Leptuca uruguayensis shows two population spawning events, with more than 65 % of the females being ovigerous. The aim of this study was to assess the energy dynamics in L. uruguayensis females to estimate how they mix the capital and the income breeding strategies in the first and second spawns of the short reproductive season. The relationship between the hepatosomatic (HIS) and gonadosomatic (GSI) indices was used to assess the contribution of the hepatopancreas to ovary maturation, as indicative of the capital breeding strategy, whereas the feeding rate and the energy available in the food resource (sediment) were estimated to assess the importance of energy intake in ovary maturation, as indicative of the income breeding strategy. Before the first spawn, a significant negative relationship between the HSI and GSI (p < 0.001) and an increase of 28.2 % in the feeding rate were recorded. This suggests that the first spawn was mainly supported by energy reserves in the hepatopancreas, indicating that females used mostly the capital breeding strategy. After the first spawn, total lipid content in the hepatopancreas decreased by 33 %. On the days before the second spawn, the relationship between the HSI and GSI showed a negative but not significant (p = 0.125) trend, whereas the feeding rate increased even more. This suggests that the intense feeding activity provided the most important energetic source for ovary re-maturation, indicating that females used mostly the income breeding strategy. Also, in the second spawn, the GSI was 24.1% smaller, a fact that affected female fecundity and weight of the egg clutches, which were respectively 12 % and 11 % lower than in the first spawn. However, the energy supply allocated to each embryo was equivalent in both spawns. This study shows how L. uruguayensis females mixed the capital and income breeding strategies to take advantage of all available resources to produce two spawns in a short reproductive season.

Losing Reproduction: Effect of High Temperature on Female Biochemical Composition and Egg Quality in a Freshwater Crustacean with Direct Development, the Red Cherry Shrimp, Neocaridina davidi (Decapoda, Atyidae).

Maternal provisioning is particularly important in invertebrates with abbreviated development because large energy reserves must be provided for the developing embryo. In this context, the objective of the present study was to analyze in an aquatic invertebrate with direct development the effect of temperature on female biochemical composition and reserve allocation to maturing ovaries, which determine egg quality. A decapod crustacean, the freshwater shrimp Neocaridina davidi, was used as experimental model. Newly hatched juveniles were exposed to 28 °C or 33 °C. Females showed mature ovaries and spawned at 28 °C (control ovigerous females), but no ovigerous female was found at 33 °C. After a 200-day period, half of the females at 33 °C were transferred to 28 °C, where they rapidly showed mature ovaries and spawned (transferred ovigerous females). Ovigerous females and females that did not spawn at 28 °C (control non-ovigerous females) and at 33 °C (high-temperature non-ovigerous females) were sacrificed to determine their biochemical composition. The number, volume, weight, and biochemical composition of the eggs from transferred and control ovigerous females were also analyzed as indicators of their quality. Female biochemical composition was not influenced by temperature, because control and high-temperature non-ovigerous females had similar lipid, protein, and glycogen contents. However, ovarian maturation and spawning were inhibited at 33 °C, which indicates a negative effect of this temperature on nutrient transfer to the oocytes. This effect was rapidly reversed after females were moved to 28 °C; the eggs from control and transferred ovigerous females were of similar quality, except for a lower protein content in the latter. The present results provide valuable information on reserve allocation to reproduction under thermal stress.

Getting ready for mating: The importance of male touching as an accelerator of ovarian growth in a caridean shrimp.

The present study was aimed at evaluating the effect of male presence on ovarian maturation in juvenile females and the role of potential chemical, visual and tactile cues emitted by males in that physiological process. A highly gregarious caridean shrimp with sexual dimorphism, Neocaridina davidi, was used as experimental model. We tested the hypothesis that male presence accelerates ovarian maturation, mainly through chemical cues. Two experiments were performed. In Experiment 1, juvenile females were reared with adult males, adult females or alone, allowing full contact among shrimps. In Experiment 2, these treatments were evaluated allowing chemical and visual communication, only visual communication, or only chemical communication among shrimps. In both experiments juvenile females were observed once a week under a stereomicroscope to determine ovarian growth rate. Although male presence was not necessary for ovarian maturation, it clearly accelerated the rate of ovarian growth, particularly in the last maturation phase. This lead to relatively longer mature ovaries with higher lipid content. On the contrary, the presence of adult females delayed ovarian maturation in juvenile females, while females reared alone showed an intermediate ovarian growth. All these results suggest that adult males release certain cues that stimulate ovarian maturation, while adult females release cues that delay this physiological process. Neither visual cues nor chemical cues released at a distance from females were responsible, either alone or in combination, for the observed effects. Ovarian growth was only influenced when shrimps were allowed to interact freely, probably because of the "mounting" behavior of males towards females. Tactile cues and/or potential chemical cues released by males during this behavior may mediate male stimulatory effect on ovarian growth. Altogether, present results partially support our initial hypothesis and contribute to increase the limited amount of information available on the role of intraspecific multimodal communication in non-behavioral reproductive processes in invertebrate species.

Energy reserves mobilization: Strategies of three decapod species.

In food deprivation assays, several different responses have been observed in crustaceans. However, studying energy reserves utilization among more than one species during the same starvation period has not yet been performed, particularly to discern whether the responses are due to intrinsic and/or environmental factors. We hypothesize that decapod species with similar feeding habits have the same strategies in the use of energetic reserves during starvation, even though they inhabit different environments. The aim of this study was to compare the energy reserves mobilization of three decapods species (Cherax quadricarinatus, Palaemon argentinus and Munida gregaria) with similar feeding habits, exposed to similar food deprivation conditions. The crayfish, shrimp and squat-lobster were experimentally kept at continuous feeding or continuous starvation throughout 15 days. Every 3rd day, the midgut gland index (MGI), and the glycogen, lipid and protein contents were measured in the midgut gland (MG) and pleon muscle. Palaemon argentinus mobilized more reserves during starvation, followed by C. quadricarinatus, and the last M. gregaria. The starved shrimps presented low MGI, whereas MG showed a reduction in glycogen (from day 6 to 15), lipid (from day 3 to 15), and protein levels (at day 9 and 15) while in their muscle, lipid reserves decreased at days 3 and 6. In C. quadricarinatus, the most affected parameters in the MG were MGI, glycogen (from day 6 to 15), and lipids (at day 12 and 15). In the MG of M. gregaria only the glycogen was reduced during fasting from 3 to 15 days. Even though the three studied species have similar feeding habitats, we found that their energetic profile utilization is different and it could be explained by the habitat, life span, temperature, organ/tissue, and metabolism of the species. Our results may be useful to understand the several different responses of crustaceans during starvation.

Long-Term Starvation and Posterior Feeding Effects on Biochemical and Physiological Responses of Midgut Gland of Cherax quadricarinatus Juveniles (Parastacidae).

We investigated the effect of long-term starvation and posterior feeding on energetic reserves, oxidative stress, digestive enzymes, and histology of C. quadricarinatus midgut gland. The crayfish (6.27 g) were randomly assigned to one of three feeding protocols: continuous feeding throughout 80 day, continuous starvation until 80 day, and continuous starvation throughout 50 day and then feeding for the following 30 days. Juveniles from each protocol were weighed, and sacrificed at day 15, 30, 50 or 80. The lipids, glycogen, reduced glutathione (GSH), soluble protein, lipid peroxidation (TBARS), protein oxidation (PO), catalase (CAT), lipase and proteinase activities, and histology were measured on midgut gland. Starved crayfish had a lower hepatosomatic index, number of molts, specific growth rate, lipids, glycogen, and GSH levels than fed animals at all assay times. The starvation did not affect the soluble protein, TBARS, PO levels and CAT. In starved juveniles the lipase activity decreased as starvation time increased, whereas proteinase activity decreased only at day 80. The histological analysis of the starved animals showed several signs of structural alterations. After 30 days of feeding, the starved-feeding animals exhibited a striking recovery of hepatosomatic index, number of molts, lipids and glycogen, GSH, lipase activity and midgut gland structure.

Female Growth and Offspring Quality over Successive Spawnings in a Caridean Shrimp Neocaridina davidi (Decapoda, Atyidae) with Direct Development.

This study analyzed the quality of recently spawned eggs and of juveniles over five and six consecutive spawns, respectively, in a caridean shrimp Neocaridina davidi with direct development. The potential energetic antagonism between reproduction and somatic growth was also evaluated. The number of eggs per spawn per female was highest in the first spawn, while the number of recently hatched juveniles per spawn per female declined in the sixth spawn. Lower lipid concentration and energy content were detected in eggs of the fourth and fifth spawns, which may indicate for the first time a decrease in maternal provisioning as a result of multiple spawning in a decapod with direct development. This result had no effect on the size of eggs or of recently hatched juveniles, nor on the growth performance of juveniles during a 30-day period following hatching. Lipids were the most abundant biochemical component of eggs, followed by proteins and glycogen; the relative proportion of each component was probably related to embryonic development type. Egg volume was unsuitable as an indicator of nutrient content, as no correlation was found between these variables. The physiological costs of reproduction were evident from the lower energy content of females that reproduced versus females that remained virgin. The lower body weight of the reproductive females at the end of the experiment showed that allocation of resources to reproduction occurred at the expense of somatic growth. To our knowledge, this is the first empirical demonstration of a decapod with direct development.

Effect of temperature on biochemical composition, growth and reproduction of the ornamental red cherry shrimp Neocaridina heteropoda heteropoda (Decapoda, Caridea).

The effect of water temperature on biochemical composition, growth and reproduction of the ornamental shrimp, Neocaridina heteropoda heteropoda, was investigated to determine the optimum temperature for its culture. The effect of embryo incubation temperature on the subsequent performance of juveniles was also evaluated. Ovigerous females and recently hatched juveniles (JI) were maintained during egg incubation and for a 90-day period, respectively, at three temperatures (24, 28 and 32 °C). Incubation period increased with decreasing water temperature, but the number and size of JI were similar among treatments. At day 30 of the 90-day period, body weight and growth increment (GI) at 24 °C were lower than those at 28 and 32 °C. On subsequent days, GI at 24 °C exceeded that at 28 and 32 °C, leading to a similar body weight among treatments. These results suggest growth was delayed at 24 °C, but only for 30 days after hatching. The lipid concentration tended to be lowest, intermediate and highest at 28, 32 and 24 °C, respectively, possibly as a consequence of the metabolic processes involved in growth and ovarian maturation. Protein and glycogen concentrations were similar among treatments. Both the growth trajectory and biochemical composition of shrimps were affected by the temperature experienced during the 90-day growth period independently of the embryo incubation temperature. During the growth period, shrimps reached sexual maturity and mated, with the highest proportion of ovigerous females occurring at 28 °C. All the females that matured and mated at 32 °C lost their eggs, indicating a potentially stressful effect of high temperature on ovarian maturation. Based on high survival and good growth performance of shrimps at the three temperatures tested over the 90-day period it is concluded that N. heteropoda heteropoda is tolerant to a wide range of water temperatures, with 28 °C being the optimum temperature for its culture.

Interference of cadmium and copper with the endocrine control of ovarian growth, in the estuarine crab Chasmagnathus granulata.

The effects of cadmium and copper on the hormonal control of ovarian growth were evaluated on the estuarine crab Chasmagnathus granulata, by means of both in vivo (14 days exposure) and in vitro (24 h) assays. For both kind of assays, heavy metal concentrations of 0 (control), 0.5 mg/L of cadmium or 0.1 mg/L of copper were used. No significant (P > 0.05) change of the gonadosomatic index was observed in the in vivo assays with intact females exposed to heavy metals, while eyestalk-ablated exposed females showed significantly (P < 0.05) lower gonadosomatic index values than their respective controls. This latter result led us to consider the possibility that the interfered with extra-eyestalk hormones. In this sense, no differences were noted between control and heavy metals-exposed groups after co-incubating ovary with thoracic ganglion (the source of the gonad stimulating hormone). However, when ovary was incubated with methyl farnesoate or 17-hydroxyprogesterone, 3H-leucine incorporation was significantly lower in the heavy metals-exposed groups than in the controls, indicating a possible interference of cadmium and copper with the transduction pathway of those hormones. On the other hand, ovaries co-incubated in vitro with eyestalk tissue and exposed to either heavy metal showed significantly higher 3H-leucine incorporation than did the controls, suggesting an inhibitory effect of both heavy metals on the secretion of the gonad inhibiting hormone from the eyestalk tissue. Interference by copper and cadmium with the transduction mechanisms of gonad inhibiting hormone at the ovarian level does not appear to be a viable hypothesis, because the addition of eyestalk extracts to the incubation medium reversed the effect caused by each heavy metal. The results from the in vitro assays were in accordance with those obtained with the intact crabs in vivo.

Toxicity, freezing and cold storage tests of native species semen / Testes de toxicidade, congelamento e refrigeração em sêmen de espécie nativa

In the first part of this study, toxicity tests were performed on the sperm of Macrobrachium acanthurus using four cryoprotectants for periods of 10 and 20 min at concentrations of 10 and 20%. In the second part, cryopreservation was performed by applying the least toxic cryoprotectant, and two freezing methods were tested over 24 hours: automated (protocols A and B) and conventional (protocols C and D). Protocol A exhibited a cooling rate of 0.5°C min-1 from -6°C to -32°C; protocol B was similar to A except for the starting temperature, which was room temperature; whereas protocols C and D exhibited a cooling rate of 2 and 10°C min-1, respectively. The third part of the study was conducted to assess the lifespan of the sperm when stored at 5°C, in which sperm viability was evaluated by a semen smear with eosin-nigrosin. The least toxic cryoprotectants were 10 and 20% glycerol, and 10% methanol, and the equilibrium time was 10 minutes. The optimal cooling rate was 2°C min-1 for 10% glycerol, which had a sperm survival rate of 21.8%. Cold storage for up to 3 days is recommended, presenting a sperm survival rate of 35.3%.(AU)

A primeira estapa deste trabalho testou a toxicidade de quatro crioprotetores em espermatozoides de Macrobrachium acanthurus durante 10 e 20 min nas concentrações de 10 e 20%. A segunda etapa foi realizar a criopreservação aplicando o crioprotetor com menor grau de toxicidade, testanto dois mecanismos de congelamento, um automatizado (protocolos A e B) e outro convencional (protocolos C e D), durante 24 horas. O protocolo A apresentou velocidade de resfriamento de 0,5°C min-1 até alcançar -32°C, partindo de uma temperatura de -6°C e idem o protocolo B, com a diferença de partir de uma temperatura ambiente; os protocolos C e D apresentaram uma velocidade de resfriamento de 2 e 10°C min-1, respectivamente, sendo as palhetas transferidas ao nitrogênio líquido. A terceira etapa foi verificar o tempo de vida do espermatozoide quando refrigerado a 5°C. A viabilidade espermática foi avaliada por meio do esfregaço de sêmen com eosina-nigrosina. Os crioprotetores que se apresentaram menos tóxicos foram o glicerol 10 e 20% e metanol 10%, num tempo de equilíbrio de 10 minutos. A melhor velocidade de congelamento foi a de 2°C min-1 para glicerol 10%, com 21,8% de sobrevivência espermática, sendo a refrigeração até três dias recomendada, com uma sobrevivência de 35,3%.(AU)

Toxicity, freezing and cold storage tests of native species semen / Testes de toxicidade, congelamento e refrigeração em sêmen de espécie nativa

In the first part of this study, toxicity tests were performed on the sperm of Macrobrachium acanthurus using four cryoprotectants for periods of 10 and 20 min at concentrations of 10 and 20%. In the second part, cryopreservation was performed by applying the least toxic cryoprotectant, and two freezing methods were tested over 24 hours: automated (protocols A and B) and conventional (protocols C and D). Protocol A exhibited a cooling rate of 0.5°C min-1 from -6°C to -32°C; protocol B was similar to A except for the starting temperature, which was room temperature; whereas protocols C and D exhibited a cooling rate of 2 and 10°C min-1, respectively. The third part of the study was conducted to assess the lifespan of the sperm when stored at 5°C, in which sperm viability was evaluated by a semen smear with eosin-nigrosin. The least toxic cryoprotectants were 10 and 20% glycerol, and 10% methanol, and the equilibrium time was 10 minutes. The optimal cooling rate was 2°C min-1 for 10% glycerol, which had a sperm survival rate of 21.8%. Cold storage for up to 3 days is recommended, presenting a sperm survival rate of 35.3%.

A primeira estapa deste trabalho testou a toxicidade de quatro crioprotetores em espermatozoides de Macrobrachium acanthurus durante 10 e 20 min nas concentrações de 10 e 20%. A segunda etapa foi realizar a criopreservação aplicando o crioprotetor com menor grau de toxicidade, testanto dois mecanismos de congelamento, um automatizado (protocolos A e B) e outro convencional (protocolos C e D), durante 24 horas. O protocolo A apresentou velocidade de resfriamento de 0,5°C min-1 até alcançar -32°C, partindo de uma temperatura de -6°C e idem o protocolo B, com a diferença de partir de uma temperatura ambiente; os protocolos C e D apresentaram uma velocidade de resfriamento de 2 e 10°C min-1, respectivamente, sendo as palhetas transferidas ao nitrogênio líquido. A terceira etapa foi verificar o tempo de vida do espermatozoide quando refrigerado a 5°C. A viabilidade espermática foi avaliada por meio do esfregaço de sêmen com eosina-nigrosina. Os crioprotetores que se apresentaram menos tóxicos foram o glicerol 10 e 20% e metanol 10%, num tempo de equilíbrio de 10 minutos. A melhor velocidade de congelamento foi a de 2°C min-1 para glicerol 10%, com 21,8% de sobrevivência espermática, sendo a refrigeração até três dias recomendada, com uma sobrevivência de 35,3%.