Starvation Promotes Autophagy-Associated Maturation of the Testis in the Giant Freshwater Prawn, Macrobrachium rosenbergii.

Autophagy is a degradative process of cellular components accomplished through an autophagosomal-lysosomal pathway. It is an evolutionary conserved mechanism present in all eukaryotic cells, and it plays a fundamental role in maintaining tissue homeostasis both in vertebrates and invertebrates. Autophagy accompanies tissue remodeling during organ differentiation. Several autophagy-related genes and proteins show significant upregulations following nutrient shortage (i.e., starvation). In our previous study, we found that in female giant freshwater prawns subjected to a short period of starvation autophagy was up-regulated in consonant with ovarian maturation and oocyte differentiation. Whether and how starvation-induced autophagy impacts on testicular maturation and spermatogenesis of the male prawns remained to be investigated. In this study, we analyzed the effects of starvation on histological and cellular changes in the testis of the giant freshwater prawn Macrobrachium rosenbergii that paralleled the induction of autophagy. Under short starvation condition, the male prawns showed increased gonado-somatic index, increased size, and late stage of maturation of seminiferous tubules, which contained increased number of spermatozoa. Concurrently, the number of autophagy vacuoles and autophagy flux, as monitored by transmission electron microscopy and the autophagic marker LC3, increased in the testicular cells, indicating that a short period of starvation could induce testicular maturation and spermatogenesis in male M. rosenbergii along with modulation of autophagy.

Autophagy-Associated Shrinkage of the Hepatopancreas in Fasting Male Macrobrachium rosenbergii Is Rescued by Neuropeptide F.

Invertebrate neuropeptide F-I (NPF-I), much alike its mammalian homolog neuropeptide Y, influences several physiological processes, including circadian rhythms, cortical excitability, stress response, and food intake behavior. Given the role of autophagy in the metabolic stress response, we investigated the effect of NPF-1 on autophagy during fasting and feeding conditions in the hepatopancreas and muscle tissues of the male giant freshwater prawn Macrobrachium rosenbergii. Starvation up-regulated the expression of the autophagy marker LC3 in both tissues. Yet, based on the relative levels of the autophagosome-associated LC3-II isoform and of its precursor LC3-I, the hepatopancreas was more responsive than the muscle to starvation-induced autophagy. Injection of NPF-I inhibited the autophagosome formation in the hepatopancreas of fasting prawns. Relative to the body weight, the muscle weight was not affected, while that of the hepatopancreas decreased upon starvation and NPF-1 treatment could largely prevent such weight loss. Thus, the hepatopancreas is the reserve organ for the nutrient homeostasis during starvation and NPF-I plays a crucial role in the balancing of energy expenditure and energy intake during starvation by modulating autophagy.

Starvation Promotes Autophagy-Associated Maturation of the Ovary in the Giant Freshwater Prawn, Macrobrachium rosenbergii.

Limitation of food availability (starvation) is known to influence the reproductive ability of animals. Autophagy is a lysosomal driven degradation process that protects the cell under metabolic stress conditions, such as during nutrient shortage. Whether, and how starvation-induced autophagy impacts on the maturation and function of reproductive organs in animals are still open questions. In this study, we have investigated the effects of starvation on histological and cellular changes that may be associated with autophagy in the ovary of the giant freshwater prawn, Macrobachium rosenbergii. To this end, the female prawns were daily fed (controls) or unfed (starvation condition) for up to 12 days, and the ovary tissue was analyzed at different time-points. Starvation triggered ovarian maturation, and concomitantly increased the expression of autophagy markers in vitellogenic oocytes. The immunoreactivities for autophagy markers, including Beclin1, LC3-II, and Lamp1, were enhanced in the late oocytes within the mature ovaries, especially at the vitellogenic stages. These markers co-localized with vitellin in the yolk granules within the oocytes, suggesting that autophagy induced by starvation could drive vitellin utilization, thus promoting ovarian maturation.

Transcriptomic analysis of the autophagy machinery in crustaceans.

BACKGROUND: The giant freshwater prawn, Macrobrachium rosenbergii, is a decapod crustacean that is commercially important as a food source. Farming of commercial crustaceans requires an efficient management strategy because the animals are easily subjected to stress and diseases during the culture. Autophagy, a stress response process, is well-documented and conserved in most animals, yet it is poorly studied in crustaceans. RESULTS: In this study, we have performed an in silico search for transcripts encoding autophagy-related (Atg) proteins within various tissue transcriptomes of M. rosenbergii. Basic Local Alignment Search Tool (BLAST) search using previously known Atg proteins as queries revealed 41 transcripts encoding homologous M. rosenbergii Atg proteins. Among these Atg proteins, we selected commonly used autophagy markers, including Beclin 1, vacuolar protein sorting (Vps) 34, microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B), p62/sequestosome 1 (SQSTM1), and lysosomal-associated membrane protein 1 (Lamp-1) for further sequence analyses using comparative alignment and protein structural prediction. We found that crustacean autophagy marker proteins contain conserved motifs typical of other animal Atg proteins. Western blotting using commercial antibodies raised against human Atg marker proteins indicated their presence in various M. rosenbergii tissues, while immunohistochemistry localized Atg marker proteins within ovarian tissue, specifically late stage oocytes. CONCLUSIONS: This study demonstrates that the molecular components of autophagic process are conserved in crustaceans, which is comparable to autophagic process in mammals. Furthermore, it provides a foundation for further studies of autophagy in crustaceans that may lead to more understanding of the reproduction- and stress-related autophagy, which will enable the efficient aquaculture practices.

The effects of serotonin, dopamine, gonadotropin-releasing hormones, and corazonin, on the androgenic gland of the giant freshwater prawn, Macrobrachium rosenbergii.

Neurotransmitters and neurohormones are agents that control gonad maturation in decapod crustaceans. Of these, serotonin (5-HT) and dopamine (DA) are neurotransmitters with known antagonist roles in female reproduction, whilst gonadotropin-releasing hormones (GnRHs) and corazonin (Crz) are neurohormones that exercise both positive and negative controls in some invertebrates. However, the effects of these agents on the androgenic gland (AG), which controls testicular maturation and male sex development in decapods, via insulin-like androgenic gland hormone (IAG), are unknown. Therefore, we set out to assay the effects of 5-HT, DA, l-GnRH-III, oct-GnRH and Crz, on the AG of small male Macrobrachium rosenbergii (Mr), using histological studies, a BrdU proliferative cell assay, immunofluorescence of Mr-IAG, and ELISA of Mr-IAG. The results showed stimulatory effects by 5-HT and l-GnRH-III through significant increases in AG size, proliferation of AG cells, and Mr-IAG production (P<0.05). In contrast, DA and Crz caused inhibitory effects on the AG through significant decreases in AG size, proliferation of AG cells, and Mr-IAG production (P<0.05). Moreover, the prawns treated with Crz died before day 16 of the experimental period. We propose that 5-HT and certain GnRHs can be now used to stimulate reproduction in male M. rosenbergii, as they induce increases in AG and testicular size, IAG production, and spermatogenesis. The mechanisms by which these occur are part of our on-going research.