Ultrastructural changes in Marsupenaeus japonicus spermatozoa in the journey from the testis to the female thelycum

Marsupenaeus japonicus is one of the most important penaeid shrimp farmed in China. As a typical closed thelycum penaeid shrimp, the morphological changes in the spermatozoa during the journey from the testis to the female thelycum remain unclear in this species. Herein, an ultrastructural comparison of the spermatozoa in the testis (TE), vas deferens (VD), and the spermatophore in the male terminal ampoule (SA) and the female seminal receptacle (SR) of M. japonicus was conducted. The spermatozoa in the VD, SA and SR shared typical morphological characteristics of the spermatozoa of decapod dendrobranchiates, including a spherical main body and a slender spike. However, no spike was observed in the spermatozoa collected from the TE. The spike of the spermatozoa in the SA and SR was longer than that in the VD, whereas no significant difference in spike length was observed between the spermatozoa from the SA and SR. The basic ultrastructure of the spermatozoa in the VD, SA and SR was similar, except for some differences in the nuclear region. The main body of the spermatozoa comprised the cytoplasmic band, nuclear region, several small and large vesicles, and the basal region of the acrosomal structure. The nuclear materials in the spermatozoa from the TE were more electron‐dense than those in the VD, SA and SR, while the nuclear region of the spermatozoa was more electron‐lucent in the SR than in the VD and SA. These findings preliminarily reveal the ultrastructural changes in M. japonicus spermatozoa during the journey from the testis to the female thelycum and provided a theoretical foundation for developing effective artificial insemination methods in this species.     

Acrosome reaction in the sperm of the black tiger shrimp Penaeus monodon (Decapoda, Penaeidae)

Owing to the problem of male infertility in the domesticated shrimp Penaeus monodon, this study was conducted to reveal the morphological events of an acrosome reaction (AR) of sperm of this highly valuable species. The AR observed in an in vitro incubation of sperm with egg water (EW) and that during actual spawning was compared. Under transmission and scanning electron microscopy, sperm taken from the female thelycum was composed of a posterior main body, a central cap and an anterior single spike. Upon contact with EW, the sperm underwent two phases of AR: acrosomal exocytosis and spherical mass formation. The former was composed of a degeneration of the spike, swelling of the cap region and rupture of the acrosomal pouch. The latter began with polymerization of materials within the subacrosomal region and ended with re‐configuration of the subacrosomal region into an electron‐dense spherical mass. The AR of the sperm observed during spawning revealed similar morphological events, with degeneration of the spike upon contact with the vitelline envelope and formation of the spherical mass while penetrating into jelly material produced by protruding cortical rods. The results suggest the presence of AR inducers derived from the vitelline envelope and cortical rods of the egg. This study forms the basis for an evaluation of infertility regarding to AR in the domesticated P. monodon male.     

鹰爪虾与中国对虾雌虾纳精囊的形态结构

使用激光扫描共聚焦显微镜对鹰爪虾与中国对虾雌虾纳精囊的形态结构进行研究。结果表明,鹰爪虾与中国对虾都具有封闭式纳精囊,鹰爪虾纳精囊表面被前两片甲壳板所覆盖,两板中间为一开口,交配后被胶体物质堵塞,纳精囊中部为单一的囊腔,囊腔向后部两侧延伸形成两个袋状的囊,为贮存精子囊的部位,中国对虾的精囊内为单一的囊腔,外面覆盖有两片圆型,左右对称的甲壳质薄片,囊腔内部由前向后伸出三个锥状的甲壳质突起,交配的雌虾     

Use of biofloc technology during the pre‐maturation period of Litopenaeus vannamei males: effect of feeds with different protein levels on the spermatophore and sperm quality

The objectives of this study were: (1) Compare two systems for pre‐maturation of Litopenaeus vannamei in terms of spermatophore and sperm quality, (2) Compare the effect of feeds with different protein levels on reproductive quality of males reared in a biofloc‐dominated system. Animals (36.40 ± 3.13 g) reared under biofloc technology (BFT) were used in the 30‐day experiment, which involved four treatments: one in a clear water system (CW) and other three in a BFT system. The BFT treatments were differentiated by feed: mix of fish, squid and crab (BFT+FF) composed of 68.48% dietary protein (DP); broodstock feed (BFT+BF) composed of 52.51% DP; and juvenile feed (BFT+JF) composed of 39.91% DP. Feed in the CW was also the mix of fresh food. Spermatophore and sperm quality were analyzed at the beginning and end of the experiment. Higher normal sperm rate was recorded in the CW compared with the BFT+FF. Among the BFT treatments, the BFT+FF had the lowest normal sperm rate. Thus, the use of BFT for pre‐maturation of L. vannamei allowed the reduction in dietary protein levels from 68.48% (BFT+FF) to 39.91% (BFT+JF) and the maintenance of spermatophore and sperm quality compared to the system based on high daily exchange rate.     

The effect of Ibuprofen on female and male reproduction of the open thelyca marine shrimp,Litopenaeus

A research was designed to explore the effect of ibuprofen (IBU) as an intervening agent for prostaglandin biosynthesis on male and female reproduction of three species of Litopenaeus. Ovarian maturation was studied in L. stylirostris and L. vannamei injected periodically with IBU at 0.01 and 0.1 μg g^?1 body weight (b.w.) and treated for unilateral eyestalk ablation. IBU at 0.1 μg induced a statistically (P < 0.05) higher rate of L. vannamei females with developing ovaries (45 ± 19%) as compared with controls (12 ± 8%) during 5 weeks; L. stylirostris did not activate vitellogenesis under this experimental protocol. IBU was also evaluated for male sexuality in L. occidentalis, L. stylirostris and L. vannamei. Periodic injections of IBU had no effect on sperm counts and spermatophore weights; however, sperm abnormalities in IBU 0.1 μg treated L. occidentalis and L. stylirostris were very low. In L. stylirostris, the renovation of normal spermatophores was remarkably improved in IBU treated males as compared with controls, but in L. vannamei spermatophore deterioration was not affected. The findings of this research support the hypothesis that prostaglandins are involved in penaeid reproduction playing a negative physiological role; however, altered steroidogenic pathways could also be involved.     

Hollow sperm syndrome during spermatogenesis in the giant tiger shrimp Penaeus monodon (Fabricius 1798) from eastern Australia

During spermatogenesis, giant tiger shrimp (Penaeus monodon) from Queensland, eastern Australia had a high proportion of testicular spermatids that appeared ‘hollow’ because their nuclei were not visible with the haematoxylin and eosin stain. When examined by transmission electron microscopy, the nuclei of hollow spermatids contained highly decondensed chromatin, with large areas missing fibrillar chromatin. Together with hollow spermatids, testicular pale enlarged (PE) spermatids with weakly staining and marginated chromatin were observed. Degenerate‐eosinophilic‐clumped (DEC) spermatids that appeared as aggregated clumps were also present in testes tubules. Among 171 sub‐adult and adult P. monodon examined from several origins, 43% displayed evidence of hollow spermatids in the testes, 33% displayed PE spermatids and 15% displayed DEC spermatids. These abnormal sperm were also found at lower prevalence in the vas deferens and spermatophore. We propose ‘Hollow Sperm Syndrome (HSS)’ to describe this abnormal sperm condition as these morphological aberrations have yet to be described in penaeid shrimp. No specific cause of HSS was confirmed by examining either tank or pond cultured shrimp exposed to various stocking densities, temperatures, salinities, dietary and seasonal factors. Compared with wild broodstock, HSS occurred at higher prevalence and severity among sub‐adults originating from farms, research ponds and tanks. Further studies are required to establish what physiological, hormonal or metabolic processes may cause HSS and whether it compromises the fertility of male P. monodon.     

Dietary Effects on Sperm Quality of Litopenaeus vannamei Broodstock

A 56‐d feeding trial was conducted to investigate the effect of diet on sperm quality of Pacific white shrimp Litopenaeus vannamei broodstock. Dietary treatments consisted of a combination of 75% dry maturation diet and 25% fresh‐frozen squid (dry‐weight basis). Supplemental nutrients of the maturation diet were selectively deleted and replaced with wheat starch to produce the following treatments: 1) 75% basal maturation diet plus 25% squid (control); 2) 75% maturation diet without supplemental vitamins plus 25% squid; 3) 75% maturation diet without supplemental cholesterol and phospholipids plus 25% squid; 4) 75% maturation diet without supplemental astaxanthin plus 25% squid; and 5) a fresh diet composed of 60% squid and 40% Maine bloodworms. Shrimp fed the control diet and the diet without supplemental astaxanthin had significantly higher mean (± SEM) change in sperm count (4.6 ± 3.2 million sperm cells and 2.9 ± 2.5 million sperm cells, respectively), with respect to baseline (8.7 ± 1.0, 6.4 ± 1.0, 9.0 ± 1.3, 6.6 ± 0.7, and 6.0 ± 0.8 million sperm cells for treatments 1, 2, 3, 4, and 5, respectively), than shrimp fed the diet without supplemental vitamins (‐1.7 ± 2.6 million sperm cells), but not significantly higher than those of shrimp receiving the diets without supplemental cholesterol‐phospholipids (1.2 ± 2.5 million sperm cells) and the fresh diet (1.3 ± 1.6 million sperm cells). Dietary deficiencies also were reflected in weight gain of shrimp fed the diet without supplemental vitamins (‐2.0 g) and the fresh diet (‐0.8 g). which were significantly lower than weight gain of shrimp fed the control diet (1.1 g) and the diet without supplemental cholesterol‐phospholipids (0.8 g). No significant differences were detected among treatments for percentage of abnormal sperm and survival data. Results demonstrated a significant effect of diet on reproductive quality of male L. vannamei and indicated that the typical combination of fresh‐food organisms used is not nutritionally optimal for male broodstock.     

Effects of the probiotic Bacillus subtilis (BP11 and BS11) on the growth and survival of Pacific white shrimp,Litopenaeus vannamei

The effect of Bacillus S11 (BS11)‐ and/or Bacillus P11 (BP11)‐supplemented feeds on the growth performance, survival, immunoenhancement and disease resistance of cultured Pacific white shrimp, Litopenaeus vannamei, was evaluated. Four feeding treatments of (i) regular feed (control), (ii) BS11‐supplemented feed, (iii) BP11‐supplemented feed and (iv) BS11‐and BP11‐supplemented feed were prepared and used for shrimp cultivation in closed recirculating cement tanks (~400 L) in two trials, one for juvenile and PL‐30 shrimp at 60 and 90 days, respectively. The results showed that BS11 gave a higher probiotic potential than BP11 for both age groups of L. vannamei in cultivation, because the average weight and survival of shrimp fed BS11‐supplemented feed were significantly higher (P < 0.05) than those of the control and the other two groups. The survival of shrimp fed either BS11‐or both BS11‐and BP11‐supplemented feed was significantly higher (P < 0.05) than that of the control group. In addition, the highest total haemocyte and granular haemocyte counts and phenoloxidase activity were found in shrimp fed with the BS11‐supplemented feed. After challenge with Vibrio harveyi 639 (~10⁷ CFU mL^?1) by immersion, the lowest cumulative death (%) and disease resistance were clearly found in shrimp fed with the BS11‐supplemented feed.     

Sperm Quality Over Consecutive Spermatophore Regenerations in the Pacific White Shrimp Litopenaeus vannamei

The reproductive potential of males plays an important role in the productivity of captive penaeids. Males present a large variability in their reproductive potential, and thus the selection of appropriate males for reproduction is highly desirable. The present study compares sperm quality at the beginning of the experiment (baseline values) with variations in sperm quality as a result of consecutive spermatophore regenerations. This was done to evaluate possible predictive criteria for optimal sperm quality based on morphological and biochemical criteria in pond-reared Pacific white shrimp Litopenaeus vannamei males. Sperm quality was similar between left and right spermatophore. Sperm count and proportion of normal sperm increased by 86 and 65% (P < 0.05) respectively, from baseline values to third spermatophore regeneration. Proportion of dead sperm progressively decreased with consecutive regenerations, attaining values 33% lower by the third regeneration, compared to baseline values. This indicates that no decrease in sperm quality occurs in L. vannamei during consecutive regenerations and that the existing spermatophores at stocking of males should be expelled to have higher sperm quality in regenerated spermatophores. Furthermore, this procedure allows selecting individuals with high baseline sperm quality based on significant correlations between baseline and regenerated sperm quality observed in the present work. Baseline values of glucose concentration in the hemolymph were positively correlated to baseline values of sperm count (r = 03, P < 0.05). In contrast, baseline values of several lipids in the hemolymph were negatively correlated to several traits of sperm quality at first sampling (baseline values) or at first regeneration. These results are discussed in terms of nutritional requirements of males and of possible predictive criteria of sperm quality.     

Sperm Quality of Pond-Reared and Wild-Caught Penaeus monodon in Thailand1

Sperm quality, as determined by visual examination and by reaction with “egg-water” was not significantly different (P > 0.05) for sperm obtained by electro-ejaculation from ablated or non-ablated pond-reared Penaeus monodon. Nor was sperm quality different between pond-reared and wild-caught prawns. Normal sperm, determined by appearance, ranged from 17.1 to 21.0%, while reactive sperm ranged from 1.5 to 3.0%. There were, however, significant correlations (P < 0.01) between spermatophore weight and prawn weight (r= 0.73, N= 434). Male prawns weighing 4150 g produced on average spermatophores weighing 22.7 mg and containing 0.8 million sperm, while prawns weighing 61-90 g produced on average spermatophores weighing 56.6 mg with 2.5 million sperm. Ablation did not increase spermatophore size or sperm quality, although it significantly increased mortality of ablated males. Male prawns could be re-ejaculated at about weekly intervals with no change in sperm quality. Wild-caught female prawns artificially inseminated with spermatophores from electro-ejaculated males produced normal spawns with 51% average egg fertilization, and 41% nauplii hatch success. Nauplii hatch success following spawning increased from >60% for newly inseminated females to near zero after 30 days post-insemination, indicating spermatophore depletion and/or deteriorated sperm quality during spermatophore storage in the thelycum. The findings of the present study indicate that electro-ejaculation and artificial insemination are relatively simple and practical methods for improving captive reproduction performance of closed-thelycum prawns such as P. monodon, and that pond-reared and wild-caught males produced sperm of similar quality.     

A novel micro‐organism for removing excess ammonia‐N in seawater ponds and the effect of Cobetia amphilecti on the growth and immune parameters of Litopenaeus vannamei

To investigate the feasibility of using micro‐organisms for ammonia‐N removal, six strains were isolated from Chinese white shrimp, Fenneropenaeus chinensis, seawater culture ponds in Dongying (Shandong, China). Of these, strain DY‐01, which exhibited the highest capacity to degrade ammonia‐N, removed 61.7% of the total ammonia‐N (50 mg/L) in 8 hr. An investigation of the factors affecting the removal efficiency indicated the optimum conditions to be 30°C, pH 8.0, and a salinity level of 30 g/L; 16S rDNA gene sequencing and biochemical analysis identified strain DY‐01 as Cobetia amphilecti, which has not previously been reported to degrade ammonia‐N. This strain also boosted the growth of Pacific white shrimp, Litopenaeus vannamei (p < .05), at a concentration of 10⁷ CFU/mL, with no harmful effects on the shrimp's immune system. This study has thus identified a novel aerobic nitrifying bacterium that is potentially an excellent candidate for improving the water quality in mariculture ponds.     

Sperm maturation and capacitation in the open thelycum shrimp Litopenaeus (Crustacea: Decapoda: Penaeoidea)

Transmission electron microscopy was applied to sperm removed from males and females belonging to Litopenaeus vannamei, L. stylirostris and L. occidentalis. It was discovered that a region named filamentous meshwork (FM), located between the nucleus and the hemispherical cap, develops differently in these three closely related species. In L. vannamei, the FM is synthesized in the male reproductive system, but seems to complete its formation after mating. In L. stylirostris, the FM region was not present in spermatophores collected from males or in sperm from the thelycum. In L. occidentalis, the FM region is fully developed in male sperm. It is suggested that completion of the FM is required for acrosome maturation, and the process continues after mating in some species of Litopenaeus. In vitro induction of the acrosome reaction in sperm from males and females of L. occidentalis demonstrated for the first time that reactivity is significantly superior in sperm cells that have been attached to the open thelycum for some hours, as compared to sperm in males (prior to transfer). This finding suggests that matured sperm cells of L. occidentalis become capacitated to react against egg water after mating.     

Effect of polysaccharides extracts of algae Ulva rigida on growth,antioxidant, immune response and resistance of shrimp,Litopenaeus vannamei against Photobacterium damselae

This study was carried out to investigate the effect of water‐soluble polysaccharides extract of algae Ulva rigida (WPU) as dietary supplement on growth performance, antioxidant enzyme activity, lysozyme and phenoloxidase activity, and resistance of shrimp (Litopenaeus vannamei) subjected to bacterial infection with Photobacterium damselae. Three replicate groups of shrimp (1.0 g) were fed four diets containing four levels, 0 or control, 0.5, 1 and 1.5 g/kg of WPU for 8 weeks over the growth trial. Thereafter, 30 shrimps from each dietary treatment were infected with bacteria P. damselae to evaluate disease resistance of infected shrimp. The results of this study showed that WPU was effective as a growth promoter for L. vannamei. The best growth rate was observed in shrimp fed 1.5 g/kg of WPU diet. Regarding antioxidant defences, the diets supplemented with three levels of WPU stimulated glutathione peroxidase and catalase activates in experimental shrimps. MDA content of L. vannamei‐fed diet containing WPU 1.5 and WPU 1.0 was lower than WPU 0 and WPU 0. 5 diets. Also, lysozyme and phenoloxidase activities of shrimp receiving WPU at 1.0 and 1.5 level were significantly higher than those fed WPU 0 and WPU 0.5 diets. In addition, using WPU extract in all diets decreased mortality in L. vannamei in a dose‐dependent manner after challenge with P. damselae. These results suggest that incorporation of water‐soluble polysaccharides from green algae U. rigida at 1.5 g/kg doses improves growth and antioxidant activity and enhances the immune responses in shrimp L. vannamei.     

Functional characterization of Farfantepenaeus californiensis, Litopenaeus vannamei and L. stylirostris haemocyte separated using density gradient centrifugation

Haemocytes from penaeid shrimp (Farfantepenaeus californiensis, Litopenaeus vannamei and L. stylirostris) were separated using a discontinuous Percoll gradient. Shrimp haemocytes were spontaneously adhered to glass, allowing slide preparations for staining and microscopic differential counting. Like other crustaceans, shrimp has three main populations differing in presence and size of cytoplasmic granules and each population seems to be biochemical or functionally compromised. Prophenoloxidase (proPO )activity was mainly located in large granules haemocytes (75%) while the small granules cells participate with 25%, but seem to be responsible for encapsulation. Haemocyte discrimination ability was tested using Sephadex? (Seph?), DEAE‐Seph? and CM‐Seph?. Only DEAE‐Seph? was encapsulated by shrimp haemocytes and provoked the release of proPO activating system, indicating the role of particle charge in the activation of shrimp immune response.     

Changes in the intestinal bacterial community during the growth of white shrimp,Litopenaeus vannamei

In this study, we documented the changes in the intestinal bacterial community at four stages in Litopenaeus vannamei: 14 days postlarvae (L14) and 1‐, 2‐ and 3‐month old juveniles (J1, J2, J3), using 454 pyrosequencing techniques. The intestinal bacterial community was dominated by three bacterial phyla, Proteobacteria, Bacteroidetes and Actinobacteria at all stages. However, the relative abundance and bacterial lineages varied at the family level. The intestinal bacterial community of L14 and J1 was similar, with dominant members belonging to the Comamonadaceae of Betaproteobacteria. Conversely, bacterial members affiliated to Flavobacteriaceae of Bacteroidetes were dominant in J2 and Vibrionaceae of Gammaproteobacteria was dominant in J3. The abundance of Microbacteriaceae of Actinobacteria also fluctuated during the four stages. Bacterial members of Flavobacteriaceae and Rhodobacteraceae (Alphaproteobacteria) were present through all growth stages, and likely form the intestinal core microbiome of L. vannamei. However, they varied at the operational taxonomic unit (OTU) level through the growth stages. The intestinal bacterial community of pond‐rearing shrimp included the three main bacterial phyla identified above, and an additional group, Mycoplasmataceae of Mollicutes. Our results demonstrate that the intestinal bacterial community of L. vannamei was highly dynamic during the growth stages. Bacterial members belonging to Commamonadaceae dominated in the earlier growth stage of shrimp, possibly influenced by feeding with Artemia nauplii, but there was a shift to Flavobacteriaceae in the mid and Vibrionaceae in the late growth stages.     

Studies of the Thermal and Haline Influences on Growth and Survival of Litopenaeus vannamei and Litopenaeus setiferus

A series of experiments were conducted to examine the effects of salinity (1–48 g/L) on the biological performance, as evaluated by growth and survival, of the Pacific white shrimp, Litopenaeus vannamei, and the Atlantic white shrimp, Litopenaeus setiferus, reared at temperatures of 20, 24 or 28 C. Poor growth and survival of L. vannamei was observed after 21–28 d of culture at low salinity (2 and 4 g/L) at 20 C. Raising salinity to 8 and up to 32 g/L significantly increased survival at this temperature, indicating that avoiding low temperatures is critical for survival of this species when reared at low salinity. A major improvement in the growth rate of L. vannamei was observed at 24 C, but it still was sub‐optimal compared to growth observed at 28 C. Irrespective of salinity, high survival rates were observed at both 24 and 28 C, but variable growth rates were recorded. Contrary to L. vannamei, the Atlantic white shrimp, L. setiferus, which was reared for 28 d at 24 C only, had better growth performance at 8 g/L compared to 2, 16 and 32 g/L. Under equal experimental conditions, L. setiferus had considerably lower weight gain and survival than L. vannamei.     

Spirulina meal has acted as a strong feeding attractant for Litopenaeus vannamei at a very low dietary inclusion level

The present work aimed at studying the growth performance and feeding preference of Litopenaeus vannamei juveniles fed on diets supplemented or not with Spirulina meal. Litopenaeus vannamei juveniles (3.89 ± 0.25 g) were stocked for 72 days in 28 round 500‐L tanks at 44 shrimp/tank (77 juveniles/m²). The diets were supplemented with 0.5% of a commercial feed attractant (C25 and C50) or with Spirulina meal (S25 and S50). In C25/S25 and C50/S50 there were reductions of 25% and 50% in fishmeal inclusion level respectively. In a further study, two feeding trays with different diets were allowed to shrimp at the same moment and they were located in opposite walls of the tank. The feed remains in each feeding tray were collected and weighted to calculate the dry feed remains. The weekly growth rate of shrimp fed on S25 (0.89 ± 0.03 g) was not significantly different from those fed on C25 (0.89 ± 0.01 g). The attractiveness experiment showed that S25 was preferred significantly more by shrimp than C25. In conclusion, Spirulina meal added at 0.5% in a complete diet for L. vannamei juveniles, with 14% of Peruvian fishmeal, has proved itself as a nutritionally efficient feeding attractant.     

Effect of vitamin E on spermatophore regeneration and quality of pond‐reared,black tiger shrimp (Penaeus monodon)

The objective of the present work was to evaluate the effects of different levels of vitamin E in feed on the spermatophore regeneration and quality of male Penaeus monodon. The experiment was carried out with the four following treatments: the basal diet no added vitamin E, the diet added 200, 600 and 1,000 mg/kg respectively. Spermatophore regeneration and quality were evaluated by spermatophore weight, sperm count and spermatophore absence rates, which male P. monodon were extruded spermatophore for feeding 20 and 40 days. In the experiment, the weight of the twice regenerated spermatophore of the males added to the vitamin E group was higher than that of the untreated control group. The weight of the first regenerated spermatophore with the addition of 1,000 mg/kg group was the highest and significantly higher than the control group (p < .05), but there was no significant difference among the three groups with different levels of vitamin E. The weight of the second regenerated spermatophore with the addition of 600 mg/kg group was the highest, followed by 1,000 mg/kg group, both of which were higher than the control group and the addition of 200 mg/kg group. Within the same group, the regeneration spermatophore weight showed overall upward trend as the feeding time, twice regenerate experiment spermatophore weight with added to the vitamin E groups were significantly higher than the initial value (p < .05), but three spermatophore weight of male shrimp at the control group had no significant difference. The sperm quantity and the percentage of normal sperm of the twice regenerated spermatophore of the males with added to the vitamin E group was higher than that of the untreated control group, and those of the addition of 200 mg/kg group was significantly higher than that of control group (p < .05). The total number of sperm and the percentage of living sperm of male shrimp in the experimental group decreased with the increase of vitamin E in the feed. Within the same group, the total number of sperm and the percentage of living sperm of male shrimp with added to the vitamin E groups showed overall upward trend as the feeding time and were significantly higher than the initial value (p < .05), but the control group was slightly down and had no significant difference. Comprehensive sperm weight, sperm quantity and living sperm percentage of three indicators, that adding 200 mg/kg of vitamin E in feed could effectively promote the spermatophore regeneration in the male P. monodon and improve the sperm quantity. The experimental results provide a scientific basis for the breeding of P. monodon.     

Viral-like particles associated with cuticular epithelium necrosis in cultured Litopenaeus vannamei (Decapoda: Crustacea) in Ecuador

A new viral agent was found associated with the endoplasmic reticulum of epithelial cells of the Pacific white shrimp Litopenaeus vannamei (Boone) sampled during mass mortalities. A 40% mortality rate affected nursery and grow‐out ponds during the first 50–60 days of culture, and peak mortality in ponds occurred when shrimp reached 2–4 g. Histopathological changes of affected shrimp showed different grades of necrosis in epithelial cells and, in some cases, other tissues were affected. Transmission electron microscopy (TEM) of columnar cells of the cuticular epithelium showed the accumulation of viral particles, either dispersed in the cytoplasm or in a string‐like or paracrystalline array. These arrays of virions were within membrane‐bound vesicles formed from the endoplasmic reticulum (ER), in orderly arrays on the outer nuclear membrane or along the ER. The virus particles had apparently proliferated in the ER. The virions had an opaque area with an approximate diameter of 20 nm and an electron‐lucent surface layer. The approximate diameter of the non‐enveloped virions was 25 nm. The cytological changes observed are similar to those associated with the Picornaviridae and Nodaviridae families. The histopathology and ultrastructure of a new disease in L. vannamei is associated with the presence of a putative new virus. Until further isolation and characterization is performed, it is recommended to refer to the agent as Litopenaeus vannamei viral‐like particles (LvVLPs).     

Transmission of white spot syndrome virus (WSSV) from Dendronereis spp. (Peters) (Nereididae) to penaeid shrimp

Dendronereis spp. (Peters) (Nereididae) is a common polychaete in shrimp ponds built on intertidal land and is natural food for shrimp in traditionally managed ponds in Indonesia. White spot syndrome virus (WSSV), an important viral pathogen of the shrimp, can replicate in this polychaete (Desrina et al. 2013); therefore, it is a potential propagative vector for virus transmission. The major aim of this study was to determine whether WSSV can be transmitted from naturally infected Dendronereis spp. to specific pathogen‐free (SPF) Pacific white shrimp Litopenaeus vannamei (Boone) through feeding. WSSV was detected in naturally infected Dendronereis spp. and Penaeus monodon Fabricius from a traditional shrimp pond, and the positive animals were used in the current experiment. WSSV‐infected Dendronereis spp. and P. monodon in a pond had a point prevalence of 90% and 80%, respectively, as measured by PCR. WSSV was detected in the head, gills, blood and mid‐body of Dendronereis spp. WSSV from naturally infected Dendronereis spp was transmitted to SPF L. vannamei and subsequently from this shrimp to new naïve‐SPF L. vannamei to cause transient infection. Our findings support the contention that Dendronereis spp, upon feeding, can be a source of WSSV infection of shrimp in ponds.