| The giant freshwater prawn(Macrobrachium rosenbergii)(De Man,1879)belongs to the Arthropoda,Crustacea,Palaemonidae,and Macrobrachium,is the largest palaemonid prawn,reaching a maximum body length of 32 cm.M.rosenbergii originated in the Indo-Pacific and was distributed to countries in Southeast Asia,including Malaysia,Thailand,India,and Bangladesh.Due to its high protein content,tasty flavor,and larger size,M.rosenbergii has become an international food source.The improvement in M.rosenbergii production has become a major research topic due to the increased market demand.Interestingly,male M.rosenbergii generally exhibit faster growth rates compared to females of similar age.The size variation in a population unfavors commercial production.Consequently,the practice of culturing monosex(all-male)prawns was developed and it is able to increase both the production yield and income.Manually selecting males for culture during the grow-out period has long been practiced in commercial prawn farming,but is labor-intensive and generally unsuccessful in producing a male monoculture.With the effort of scientists,it is found that the androgenic gland is essential for sexual differentiation,development,and maintaining the sexual morphotype features in male crustaceans.The androgenic gland ablation and the knockdown of M.rosenbergii insulin-like androgenic gland hormone(Mr IAG)and M.rosenbergii insulin-like receptor(Mr IR)may induce sex reversal,turning male to neo-female.The mating of neo-females with males will produce all male progeny which favors the monosex culture.With the emergence of transcriptomic sequencing technology and the advances in molecular and biotechnology,it facilitates the deeper understanding of sex reversal studies in M.rosenbergii.In this studies,androgenic gland ablation approach,transcriptomic profiling,small-interfering RNA(si RNA)knockdown,double-strand RNA(ds RNA)knockdown,in situ hybridization,colocalization,RT-q PCR,morphological and histological analysis were employed to elucidate the effect of androgenic gland ablation on gonad development and sex reversal,the transcription pattern of male sex-associated genes across the gonadal development in different morphotype,the localization of Mr IR in male gonad,the interaction between Mr IR and Mr IAG,the sex reversal induced by the knockdown of Mr IR and Mr IAG via ds RNA and si RNA.The main contents and results are as follows:1.Transcription profiling and insights into the effect of androgenic gland ablation on gonad development in M.rosenbergii.Transcriptomic profiling generates 42.32-42.92 million clean reads.A total of 94,747 unigenes were assembled,and total length(160,460,655 bp),average length(1,693 bp),N50(3,438 bp),and GC content(39.86 %)were obtained.After androgenic ablation,the testis undergoes degeneration and spermatogenesis is arrested,and further proceeds to ovary development.These phenomena may trigger a series of cellular processes in response to the changes that happened.It is noteworthy that Hsp70,IGFBP7,Mar-Mrr,Serpin,TUBA3,CREB,EF1 a,Bmp7 were identified as differentially expressed in the transcriptomic profiling of gonads after androgenic gland ablation.The above-mentioned genes are speculated to be vital in the regulation of gonad development,cell proliferation,protein synthesis,and acceleration of RNA transcription.Interestingly,the Rap1 signaling pathway,hippo signaling pathway,PI3 K signaling pathway,IL-17 signaling pathway,oxytocin signaling pathway,thyroid hormone signaling pathway,apoptosis signaling pathway,and ECM-receptor interaction signaling pathway were enriched in the sex reversal process,in which regulates cell proliferation,gonad development,female puberty regulation,and maintaining normal homeostasis.The sexual morphology features changes happened in male M.rosenbergii after androgenic gland ablation.The male sexual morphotype,including genital papillae,appendix interna,appendix masculine,and gonopore flap,disappeared and the female sexual morphotype,such as brood chamber,setal buds,ovigerous setae,and ovipositing setae,can be observed at 28 days after androgenic gland ablation.At the same time,histological observation revealed that the testis was degenerated,and spermatogenesis was arrested,while the ovary was initiated to develop as the previtellogenic oocytes and developing oocytes can be observed at 28 days after androgenic ablation onwards.2.Gene expression pattern across male gonads in juvenile,orange-claw,and blue-claw M.rosenbergii.The findings revealed that Mr IAG,Mr IR,Mro Sxl1,and Mro Dmrt11 E showed an increasing pattern in the testis;while MRPINK,Mar-Mrr,displayed an increasing pattern in vas deferens;and Mro Dmrt99 B showed a decreasing pattern.The Mr IAG,Mr IR,Mro Sxl1,and Mro Dmrt11 E,MRPINK,and Mar-Mrr have the highest transcription levels in the blue-claw prawn.Interestingly,the Mr IAG has an increasing pattern for both the testis and androgenic gland,and the transcription level also highest in the blue-claw prawn.The transcription pattern of Mr IAG,Mr IR,Mro Sxl1,Mro Dmrt11 e,Mar-Mrr,Mro Dmrt99 b,and MRPINK across the male gonad(testis,vas deferens,and androgenic gland)at 1 cm,orange-claw,and blue-claw stages of M.rosenbergii suggest that these genes have their key function through the projection of transcription level.It is notable that most of the genes showed the highest transcription in the blue-claw stage.This phenomenon inferred that the blue-claw stage has more reproductive events than the orange-claw.3.Localization,interaction of Mr IR with Mr IAG,and si RNA knockdown of Mr IRThe localization of Mr IR in M.rosenbergii was discovered and found that Mr IR was mainly expressed in the spermatocytes,androgenic gland cells,and secretory epithelial cells of terminal ampullae.The co-localization between Mr IR and Mr IAG speculated that Mr IR functions as a receptor for Mr IAG.The optimal injection dose of si RNA-Mr IR knockdown was 0.50 μg/g body weight and the long-term knockdown experiment yielded neo-females.Through histological observations,it is inferred that the injection dose of 0.50 μg/g body weight can efficiently retard spermatogenesis in testes.The seminiferous lobules were loosely arranged,and only spermatocytes could be observed.Morphologically,neo-females developed as female prawns that contained a brood chamber(sperm-receiving pouch),setal buds,ovipositing setae and ovigerous setae.However,the second pereiopod was longer than in the female control,but shorter than in the male control.The knockdown of Mr IR vis si RNA with a dose of 0.50 μg/g body weight in male M.rosenbergii led to sex reversal from male to neo-female.This study revealed that the knockdown of Mr IR with 0.50 μg/g body weight can successfully induce sex reversal in male M.rosenbergii.4.Sex reversal via si RNA-mediated Mr IAG knockdownThis study employed si RNA approach to knockdown Mr IAG in male M.rosenbergii.In the current study,the optimal injection dosage to achieve sex reversal was 0.50 μg/g body weight.After Mr IAG knockdown,the expression levels of Dmrt11 e,Dmrt99b,MRPINK,Mrr,Sxl1 and Sxl2 decreased significantly.With long-term knockdown effect on Mr IAG,the ds RNA and si RNA approaches produce neo-females.The neo-female has a wider brood chamber,ovipositing setae and ovigerous setae which resemble normal females.After a long-term knockdown with si RNA,most of the germ cells were arrested at the spermatocyte stage,but the spermatocytes in control could further develop into spermatozoon.The seminiferous tubules are loosely arranged,and the spermatocytes are more than spermatozoon in the 0.50 μg/g body weight treatment dose.The knockdown of Mr IAG via si RNA with a dose of 0.50 μg/g body weight in male M.rosenbergii led to sex reversal from male to neo-female. |
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