Craig L. Browdy

Immune gene discovery by expressed sequence tag analysis of hemocytes and hepatopancreas in the Pacific White Shrimp, Litopenaeus vannamei, and the Atlantic White Shrimp, L. setiferus.

A pilot program was undertaken in immune gene discovery in two sister species of litopenaeid shrimp, the Pacific white shrimp, Litopenaeus vannamei and the Atlantic white shrimp, L. setiferus. RNA from the hemocytes and hepatopancreas of single individuals from each species was recovered, 4 cDNA libraries (one from each tissue/species) were made by a PCR-based method and a total of approximately 2045 randomly selected clones were sequenced. A total of 268 expressed sequence tags (ESTs) were found that corresponded to 44 immune function genes. The most common immune-function ESTs (172) were antimicrobial peptides, which were restricted to the hemocyte libraries. Lectins were the largest group of immune-function ESTs found in the hepatopancreas. Analysis of these libraries indicates that EST approaches are effective for immune gene discovery in shrimp and that the diversity of these PCR-generated libraries would support full-scale EST collection.

Induction of Antiviral Immunity by Double-Stranded RNA in a Marine Invertebrate

ABSTRACT Vertebrates mount a strong innate immune response against viruses, largely by activating the interferon system. Double-stranded RNA (dsRNA), a common intermediate formed during the life cycle of many viruses, is a potent trigger of this response. In contrast, no general inducible antiviral defense mechanism has been reported in any invertebrate. Here we show that dsRNA induces antiviral protection in the marine crustacean Litopenaeus vannamei. When treated with dsRNA, shrimp showed increased resistance to infection by two unrelated viruses, white spot syndrome virus and Taura syndrome virus. Induction of this antiviral state is independent of the sequence of the dsRNA used and therefore distinct from the sequence-specific dsRNA-mediated genetic interference phenomenon. This demonstrates for the first time that an invertebrate immune system, like its vertebrate counterparts, can recognize dsRNA as a virus-associated molecular pattern, resulting in the activation of an innate antiviral response.

Double-Stranded RNA Induces Sequence-Specific Antiviral Silencing in Addition to Nonspecific Immunity in a Marine Shrimp: Convergence of RNA Interference and Innate Immunity in the Invertebrate Antiviral Response?

ABSTRACT Double-stranded RNA (dsRNA) is a common by-product of viral infections and a potent inducer of innate antiviral immune responses in vertebrates. In the marine shrimp Litopenaeus vannamei, innate antiviral immunity is also induced by dsRNA in a sequence-independent manner. In this study, the hypothesis that dsRNA can evoke not only innate antiviral immunity but also a sequence-specific antiviral response in shrimp was tested. It was found that viral sequence-specific dsRNA affords potent antiviral immunity in vivo, implying the involvement of RNA interference (RNAi)-like mechanisms in the antiviral response of the shrimp. Consistent with the activation of RNAi by virus-specific dsRNA, endogenous shrimp genes could be silenced in a systemic fashion by the administration of cognate long dsRNA. While innate antiviral immunity, sequence-dependent antiviral protection, and gene silencing could all be induced by injection of long dsRNA molecules, injection of short interfering RNAs failed to induce similar responses, suggesting a size requirement for extracellular dsRNA to engage antiviral mechanisms and gene silencing. We propose a model of antiviral immunity in shrimp by which viral dsRNA engages not only innate immune pathways but also an RNAi-like mechanism to induce potent antiviral responses in vivo.

Effects of sand sediment and vertical surfaces (AquaMatsTM) on production, water quality, and microbial ecology in an intensive Litopenaeus vannamei culture system

Growth and production of Pacific white shrimp (Litopenaeus vannamei) was examined along with water quality, and general indicators of microbial activity in replicate intensive tank cultures with no sediment, sand sediment, and sand sediment together with additional vertical surfaces (sand/mat). All treatments received similar feed and fertilizer input. Initial linear shrimp growth rates was relatively high (1.65 g/week) in all treatments. A reduction in growth rates was seen in the no-sediment and sand treatments, while linear growth continued longer in the sand/mat treatment. Shrimp weight, survival and production was significantly greater in the sand/mat treatment than in the sand and no-sediment treatments. While the latter two treatments yielded similar production, the no-sediment treatment required weekly additions of soda ash during the last month of the 104-day season to maintain a minimum pH of 7.0. Significantly higher pH, in conjunction with lower phytoplankton biomass and ammonia in the sand/mat treatment than in the sand treatment suggests greater carbon fixation by the attached algal community of the sand/mat treatment. This is further supported by similar carbon mineralization rates in all treatments as roughly indicated by water respiration rates in conjunction with greater sludge accumulation in the sand/mat treatment. Phyto and bacterioplankton levels were significantly lower in the sand/mat treatment than in the other treatments. Ammonia, nitrate and sludge accumulation were similar in the no-sediment and sand tanks. The sand/mat treatment had significantly greater nitrite than the other treatments. Water column nitrification rates were similar in the sand and no-sediment treatments and significantly greater in the sand/mat treatment, suggesting that nutrient cycling rates may be greater in this treatment. From a production standpoint, the primary difference in the no-sediment and sand treatments was the need for proactive management of pH in the no-sediment tanks. The quantity of vertical surface area addition tested in the sand/mat treatment of this study yielded higher shrimp production in conjunction with significant differences in water quality and indicators of general activity, suggesting that this relatively high level of vertical surface addition may significantly affect aquaculture production systems.

Recent developments in penaeid broodstock and seed production technologies: improving the outlook for superior captive stocks

Pond culture of marine shrimp currently accounts for about 30% of world supply and production continues to expand. The future success of shrimp farming will depend upon increasing supplies of healthy, high quality seed for stocking ponds. In many areas, fry are still collected from the wild for stocking into ponds. To stabilize seed supplies and expand to new areas, hatchery production technologies have improved slowly over the past 20 years. Challenges associated with disease control and reducing reliance on natural feeds are being met through research efforts in the private and public sectors. Improved hygiene and biosecurity, development of probiotics and immunostimulants and improvement of artificial feeds promise better postlarval fitness while reducing cost and improving reliability of production. However, much of this production continues to rely on nauplii derived from wild gravid spawners collected at sea. Although techniques for captive maturation and spawning have been well defined, serious gaps remain in the basic understanding of the hormonal control of reproduction, the process of vitellogenesis in penaeid shrimp and the mechanical and physiological processes involved in fertilization. Presently, in areas with readily available indigenous stocks of species of choice, there is a generally accepted perception among hatchery managers and growers that performance of nauplii and postlarvae derived from wild matured spawners is superior to captive matured or pond reared animals. As we approach the 21st century, wild stocks are facing increasing pressure and shrimp farming continues to expand into new areas, away from traditional sources of wild seed. New viral disease epidemics continue to arise and spread quickly across entire regions, significantly impacting industry viability. Research and development efforts aimed at closing the life cycle of several commercially important species are underway. While significant limitations remain, encouraging results illustrating the potential for disease control, disease resistance and improved growth have been reported. The development of healthy, genetically improved shrimp stocks represents a tremendous economic opportunity while potentially improving the outlook for long term industry sustainability. The present paper is a review of recent developments in penaeid broodstock and seed production technologies focusing on current limitations while defining the significant potential offered by emerging developments.

Non-specific activation of antiviral immunity and induction of RNA interference may engage the same pathway in the Pacific white leg shrimp Litopenaeus vannamei.

Many questions remain unanswered regarding RNAi-based mechanisms and dsRNA-induced antiviral immune responses in penaeid shrimp. In this study, we report the characterization in the white leg shrimp Litopenaeus vannamei of RNAi pathway associated proteins Lv-Ago 1 and Lv-Ago 2, two members of the Argonaute family of proteins, as well as Lv-sid 1, the first shrimp homologue of Sid-1, a membrane channel-forming protein implicated in the cellular import of dsRNA. To decipher their functional implication in RNAi-related phenomena, we monitored their relative expression following stimulation by specific and non-specific RNA duplexes of diverse length. The findings show that the length of small RNA duplexes plays a critical role in the activation of both RNAi-related and innate antiviral responses. They also suggest that these two mechanisms of antiviral response may activate the same pathway, requiring Lv-Sid 1 and Lv-Ago 2 induction.

Identification of Bacillus Strains for Biological Control of Catfish Pathogens

Bacillus strains isolated from soil or channel catfish intestine were screened for their antagonism against Edwardsiella ictaluri and Aeromonas hydrophila, the causative agents of enteric septicemia of catfish (ESC) and motile aeromonad septicaemia (MAS), respectively. Twenty one strains were selected and their antagonistic activity against other aquatic pathogens was also tested. Each of the top 21 strains expressed antagonistic activity against multiple aquatic bacterial pathogens including Edwardsiella tarda, Streptococcus iniae, Yersinia ruckeri, Flavobacterium columnare, and/or the oomycete Saprolegnia ferax. Survival of the 21 Bacillus strains in the intestine of catfish was determined as Bacillus CFU/g of intestinal tissue of catfish after feeding Bacillus spore-supplemented feed for seven days followed by normal feed for three days. Five Bacillus strains that showed good antimicrobial activity and intestinal survival were incorporated into feed in spore form at a dose of 8×107 CFU/g and fed to channel catfish for 14 days before they were challenged by E. ictaluri in replicate. Two Bacillus subtilis strains conferred significant benefit in reducing catfish mortality (P<0.05). A similar challenge experiment conducted in Vietnam with four of the five Bacillus strains also showed protective effects against E. ictaluri in striped catfish. Safety of the four strains exhibiting the strongest biological control in vivo was also investigated in terms of whether the strains contain plasmids or express resistance to clinically important antibiotics. The Bacillus strains identified from this study have good potential to mediate disease control as probiotic feed additives for catfish aquaculture.

The role of crustins in Litopenaeus vannamei in response to infection with shrimp pathogens: An in vivo approach

Crustin antimicrobial peptides, identified in crustaceans, are hypothesized to have both antimicrobial and protease inhibitor activity based on their primary structure and in vitro assays. In this study, a reverse genetic approach was utilized to test the hypothesis that crustins are antimicrobial in vivo in response to bacterial and fungal challenge. Injection of double-stranded RNA specific to a 120-bp region of LvABP1, one of the most prominent crustin isoforms, yielded a significant reduction in the expression of both crustin mRNA and protein within the hemocytes. To test the role of crustins in the shrimp immune response, RNAi was first used to suppress crustin expression and animals were subsequently injected with low pathogenic doses of either Vibrio penaeicida or Fusarium oxysporum. A significant increase in mortality in crustin-depleted animals was observed in animals infected with V. penaeicida as compared to controls, whereas no significant change in shrimp mortality was observed following infection with F. oxysporum.

Knocking down caspase-3 by RNAi reduces mortality in Pacific white shrimp Penaeus (Litopenaeus) vannamei challenged with a low dose of white-spot syndrome virus.

Apoptosis has long been observed in viral target organs of white-spot syndrome virus (WSSV)-infected shrimp and whether the phenomenon helps the shrimp to survive the infection or is a factor leading to mortality is still controversial. If the shrimp mortality is a result of triggered apoptosis, then inactivation of caspase-3, a key protein in the induction of apoptosis, should improve shrimp survival upon challenge with WSSV. To test this prediction, we identified and characterized a caspase-3 homologue (cap-3) from the Pacific white shrimp Penaeus (Litopenaeus) vannamei and used this information to silence cap-3 expression by RNA interference prior to WSSV challenge. After confirming the efficacy of cap-3 silencing, its effects on mortality at high and low doses of WSSV were evaluated. In a high-dose WSSV challenge, cap-3 silencing had no significant effect on WSSV-induced mortality, except for a delay in mean time to death. However, at a low-dose WSSV challenge, cap-3 silencing correlated with a lower level of cumulative mortality, relative to silencing of a control gene, suggesting that apoptosis may exacerbate rather than decrease mortality in WSSV-challenged shrimp.

Contributions of functional genomics and proteomics to the study of immune responses in the Pacific white leg shrimp Litopenaeus vannamei

The need for better control of infectious diseases in shrimp aquaculture and the ecological importance of crustacea in marine ecosystems have prompted interest in the study of crustacean immune systems, particularly those of shrimp. As shrimp and other crustacea are poorly understood from the immunological point of view, functional genomic and proteomic approaches have been applied as a means of quickly obtaining molecular information regarding immune responses in these organisms. In this article, a series of results derived from transcriptomic and proteomic studies in shrimp (Litopenaeus vannamei) are discussed. Expressed Sequence Tag analysis, differential expression cloning through Suppression Subtractive Hybridization, expression profiling using microarrays, and proteomic studies using mass spectrometry, have provided a wealth of useful data and opportunities for new avenues of research. Examples of new research directions arising from these studies in shrimp include the molecular diversity of antimicrobial effectors, the role of double stranded RNA as an inducer of antiviral immunity, and the possible overlap between antibacterial and antiviral responses in the shrimp.