Skip to main content

Advances, challenges, and directions in shrimp disease control: the guidelines from an ecological perspective

Applied Microbiology and Biotechnology volume 100, pages 6947–6954 (2016)Cite this article

Abstract

High-density aquaculture has led to increasing occurrences of diseases in shrimp. Thus, it is imperative to establish effective and quantitative strategies for preventing and predicting these diseases. Water quality indices and investigations of specific pathogen abundance provide only a qualitative evaluation of the risk of shrimp disease and can be inaccurate. To address these shortcomings, we introduced intestinal indicative assemblages as independent variables with which to quantitatively predict incidences of shrimp disease. Given the ignorance regarding the niches differences in the shrimp intestine throughout its developmental stages, the use of probiotics in aquaculture has had limited success. Therefore, we propose the exploration of effective probiotic bacteria from shrimp intestinal flora and the establishment of therapeutic strategies dependent on shrimp age. Following ecological selection principles, we hypothesize that the larval stage provides the best opportunity to establish a desired gut microbiota through preemptive colonization of the treated rearing water with known probiotics. To employ this strategy, however, substantial barriers must be overcome.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2

References

  • Alavandi S, Vijayan K, Santiago T, Poornima M, Jithendran K, Ali S, Rajan J (2004) Evaluation of Pseudomonas sp. PM 11 and Vibrio fluvialis PM 17 on immune indices of tiger shrimp, Penaeus monodon. Fish Shellfish Immunol 17:115–120

    CAS  Article  PubMed  Google Scholar 

  • Alderman DJ, Hastings TS (2004) Antibiotic use in aquaculture: development of antibiotic resistance-potential for consumer health risks. Int J Food Sci Technol 33:139–155

    Article  Google Scholar 

  • Attramadal KJ, Salvesen I, Xue R, Øie G, Størseth TR, Vadstein O, Olsen Y (2012) Recirculation as a possible microbial control strategy in the production of marine larvae. Aquac Eng 46:27–39

    Article  Google Scholar 

  • Beardsley C, Moss S, Malfatti F, Azam F (2011) Quantitative role of shrimp fecal bacteria in organic matter fluxes in a recirculating shrimp aquaculture system. FEMS Microbiol Ecol 77:134–145

    CAS  Article  PubMed  Google Scholar 

  • Biao X, Kaijin Y (2007) Shrimp farming in China: operating characteristics, environmental impact and perspectives. Ocean Coast Manag 50:538–550

    Article  Google Scholar 

  • Boutin S, Bernatchez L, Audet C, Derôme N (2013) Network analysis highlights complex interactions between pathogen, host and commensal microbiota. PLoS One 8:e84772

    Article  PubMed  PubMed Central  Google Scholar 

  • Burns AR, Stephens WZ, Stagaman K, Wong S, Rawls JF, Guillemin K, Bohannan BJ (2016) Contribution of neutral processes to the assembly of gut microbial communities in the zebrafish over host development. ISME J 10:655–664

    Article  PubMed  Google Scholar 

  • Cao H, An J, Zheng W, He S (2015) Vibrio cholerae pathogen from the freshwater-cultured whiteleg shrimp Penaeus vannamei and control with Bdellovibrio bacteriovorus. J Invertebr Pathol 130:13–20

    Article  PubMed  Google Scholar 

  • Cavender-Bares J, Kozak KH, Fine PV, Kembel SW (2009) The merging of community ecology and phylogenetic biology. Ecol Lett 12:693–715

    Article  PubMed  Google Scholar 

  • Chave J (2004) Neutral theory and community ecology. Ecol Lett 7:241–253

    Article  Google Scholar 

  • Cheng W, Wang LU, Chen JC (2005) Effect of water temperature on the immune response of white shrimp Litopenaeus vannamei to Vibrio alginolyticus. Aquaculture 250:592–601

    Article  Google Scholar 

  • Cheung MK, Yip HY, Nong W, Law PTW, Chu KH, Kwan HS, Hui JHL (2015) Rapid change of microbiota diversity in the gut but not the hepatopancreas during gonadal development of the new shrimp model Neocaridina denticulata. Mar Biotechnol 17:811–819

    CAS  Article  PubMed  Google Scholar 

  • Clemente JC, Ursell LK, Parfrey LW, Knight R (2012) The impact of the gut microbiota on human health: an integrative view. Cell 148:1258–1270

    CAS  Article  PubMed  Google Scholar 

  • De Schryver P, Vadstein O (2014) Ecological theory as a foundation to control pathogenic invasion in aquaculture. ISME J 8:2360–2368

    Article  PubMed  PubMed Central  Google Scholar 

  • De Schryver P, Defoirdt T, Sorgeloos P (2014) Early mortality syndrome outbreaks: a microbial management issue in shrimp farming? PLoS Pathog 10:e1003919

    Article  PubMed  PubMed Central  Google Scholar 

  • Defoirdt T, Sorgeloos P, Bossier P (2011) Alternatives to antibiotics for the control of bacterial disease in aquaculture. Curr Opin Microbiol 14:251–258

    Article  PubMed  Google Scholar 

  • Dufrecircne M, Legendre P (1997) Species assemblages and indicator species: the need for a flexible asymmetrical approach. Ecol Monogr 67:345–366

    Google Scholar 

  • Ferreira N, Bonetti C, Seiffert W (2011) Hydrological and water quality indices as management tools in marine shrimp culture. Aquaculture 318:425–433

    Article  Google Scholar 

  • Fjellheim AJ, Playfoot KJ, Skjermo J, Vadstein O (2012) Inter-individual variation in the dominant intestinal microbiota of reared Atlantic cod (Gadus morhua L.) larvae. Aquac Res 43:1499–1508

    Article  Google Scholar 

  • Greenhalgh K, Meyer KM, Aagaard KM, Wilmes P (2016) The human gut microbiome in health: establishment and resilience of microbiota over a lifetime. Environ Microbiol. doi:10.1111/1462-2920.13318

    PubMed  Google Scholar 

  • Guarner F, Malagelada JR (2003) Gut flora in health and disease. Lancet 361:512–519

    Article  PubMed  Google Scholar 

  • Hasson KW, Wyld EM, Fan Y, Lingsweiller SW, Weaver SJ, Cheng J, Varner PW (2009) Streptococcosis in farmed Litopenaeus vannamei: a new emerging bacterial disease of penaeid shrimp. Dis Aquat Org 86:93–106

    CAS  Article  PubMed  Google Scholar 

  • Hsu S, Chen J (2007) The immune response of white shrimp Penaeus vannamei and its susceptibility to Vibrio alginolyticus under sulfide stress. Aquaculture 271:61–69

    CAS  Article  Google Scholar 

  • Huang Z, Li X, Wang L, Shao Z (2016) Changes in the intestinal bacterial community during the growth of white shrimp, Litopenaeus vannamei. Aquac Res 47:1737–1746

    Article  Google Scholar 

  • Johnson KN, van Hulten MC, Barnes AC (2008) “Vaccination” of shrimp against viral pathogens: phenomenology and underlying mechanisms. Vaccine 26:4885–4892

    CAS  Article  PubMed  Google Scholar 

  • Lathrop SK, Bloom SM, Rao SM, Nutsch K, Lio CW, Santacruz N, Peterson DA, Stappenbeck TS, Hsieh CS (2011) Peripheral education of the immune system by colonic commensal microbiota. Nature 478:250–254

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Leung PS, Tran LT (2000) Predicting shrimp disease occurrence: artificial neural networks vs. logistic regression. Aquaculture 187:35–49

    Article  Google Scholar 

  • Li K, Zheng T, Tian Y, Xi F, Yuan J, Zhang G, Hong H (2007) Beneficial effects of Bacillus licheniformis on the intestinal microflora and immunity of the white shrimp, Litopenaeus vannamei. Biotechnol Lett 29:525–530

    CAS  Article  PubMed  Google Scholar 

  • Liu B, Yu Z, Song X, Guan Y, Jian X, He J (2006) The effect of acute salinity change on white spot syndrome (WSS) outbreaks in Fenneropenaeus chinensis. Aquaculture 253:163–170

    CAS  Article  Google Scholar 

  • Lozupone CA, Stombaugh JI, Gordon JI, Jansson JK, Knight R (2012) Diversity, stability and resilience of the human gut microbiota. Nature 489:220–230

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Lucas R, Courties C, Herbland A, Goulletquer P, Marteau AL, Lemonnier H (2010) Eutrophication in a tropical pond: understanding the bacterioplankton and phytoplankton dynamics during a vibriosis outbreak using flow cytometric analyses. Aquaculture 310:112–121

    Article  Google Scholar 

  • Ma Z, Song X, Wan R, Gao L (2013) A modified water quality index for intensive shrimp ponds of Litopenaeus vannamei. Ecol Indic 24:287–293

    CAS  Article  Google Scholar 

  • Mohanty RK, Mishra A, Panda DK, Patil DU (2014) Effects of water exchange protocols on water quality, sedimentation rate and production performance of Penaeus monodon in earthen ponds. Aquac Res 46(10):2457–2468

    Article  Google Scholar 

  • Mühling M, Woolven-Allen J, Murrell JC, Joint I (2008) Improved group-specific PCR primers for denaturing gradient gel electrophoresis analysis of the genetic diversity of complex microbial communities. ISME J 2:379–392

    Article  PubMed  Google Scholar 

  • Munro J, Oakey J, Bromage E, Owens L (2003) Experimental bacteriophage-mediated virulence in strains of Vibrio harveyi. Dis Aquat Org 54:187–194

    Article  PubMed  Google Scholar 

  • Nemergut DR, Schmidt SK, Fukami T, O’Neill SP, Bilinski TM, Stanish LF, Knelman JE, Darcy JL, Lynch RC, Wickey P (2013) Patterns and processes of microbial community assembly. Microbiol Mol Biol Rev 77:342–356

    Article  PubMed  PubMed Central  Google Scholar 

  • Ninawe A, Selvin J (2009) Probiotics in shrimp aquaculture: avenues and challenges. Crit Rev Microbiol 35:43–66

    CAS  Article  PubMed  Google Scholar 

  • Olafsen JA (2001) Interactions between fish larvae and bacteria in marine aquaculture. Aquaculture 200:223–247

    Article  Google Scholar 

  • Ostrensky A, Poersch L (1992) Toxicidade aguda do nitrito na larvicultura do camarão-rosa Penaeus paulensis Pérez-Farfante, 1967. Nerítica 7:101–107

    Google Scholar 

  • Oxley AP, Shipton W, Owens L, McKay D (2002) Bacterial flora from the gut of the wild and cultured banana prawn, Penaeus merguiensis. J Appl Microbiol 93:214–223

    CAS  Article  PubMed  Google Scholar 

  • Páez-Osuna F (2001) The environmental impact of shrimp aquaculture: causes, effects and mitigating alternatives. Environ Manag 28:131–140

    Article  Google Scholar 

  • Pérez T, Balcázar J, Ruiz-Zarzuela I, Halaihel N, Vendrell D, de Blas I, Múzquiz J (2010) Host-microbiota interactions within the fish intestinal ecosystem. Mucosal Immunol 3:355–360

    Article  PubMed  Google Scholar 

  • Rengpipat S, Tunyanun A, Fast AW, Piyatiratitivorakul S, Menasveta P (2003) Enhanced growth and resistance to Vibrio challenge in pond-reared black tiger shrimp Penaeus monodon fed a Bacillus probiotic. Dis Aquat Org 55:169–173

    Article  PubMed  Google Scholar 

  • Rittmann BE, Martina H, Frank LF, Love NG, Gerard M, Satoshi O, Oerther DB, Jordan P, Lutgarde R, Michael W (2006) A vista for microbial ecology and environmental biotechnology. Environ Sci Technol 40:1096–1103

    Article  PubMed  Google Scholar 

  • Roeselers G, Mittge EK, Stephens WZ, Parichy DM, Cavanaugh CM, Guillemin K, Rawls JF (2011) Evidence for a core gut microbiota in the zebrafish. ISME J 5:1595–1608

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Rogers GB, Hoffman LR, Carroll MP, Bruce KD (2013) Interpreting infective microbiota: the importance of an ecological perspective. Trends Microbiol 21:271–276

    CAS  Article  PubMed  Google Scholar 

  • Rosenberry B (2003) World shrimp farming 2003. Shrimps News International

  • Round JL, Mazmanian SK (2009) The gut microbiota shapes intestinal immune responses during health and disease. Nat Rev Immunol 9:313–323

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Rungrassamee W, Klanchui A, Chaiyapechara S, Maibunkaew S, Tangphatsornruang S, Jiravanichpaisal P, Karoonuthaisiri N (2013) Bacterial population in intestines of the black tiger shrimp (Penaeus monodon) under different growth stages. PLoS One 8:e60802

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Rungrassamee W, Klanchui A, Maibunkaew S, Chaiyapechara S, Jiravanichpaisal P, Karoonuthaisiri N (2014) Characterization of intestinal bacteria in wild and domesticated adult black tiger shrimp (Penaeus monodon). PLoS One 9:e91853

    Article  PubMed  PubMed Central  Google Scholar 

  • Rungrassamee W, Klanchui A, Maibunkaew S, Karoonuthaisiri N (2016) Bacterial dynamics in intestines of the black tiger shrimp and the Pacific white shrimp during Vibrio harveyi exposure. J Invertebr Pathol 133:12–19

    CAS  Article  PubMed  Google Scholar 

  • Sandi W, Rawls JF (2012) Intestinal microbiota composition in fishes is influenced by host ecology and environment. Mol Ecol 21:3100–3102

    Article  Google Scholar 

  • Schmidt VT, Smith KF, Melvin DW, Amaral-Zettler LA (2015) Community assembly of a euryhaline fish microbiome during salinity acclimation. Mol Ecol 24:2537–2550

    Article  PubMed  Google Scholar 

  • Shade A, Peter H, Allison SD, Baho DL, Berga M, Bürgmann H, Huber DH, Langenheder S, Lennon JT, Martiny JB, Matulich KL, Schmidt TM, Handelsman J (2012) Fundamentals of microbial community resistance and resilience. Front Microbiol 3:417

    Article  PubMed  PubMed Central  Google Scholar 

  • Solanki HG, Bhatt JH, Gopal C, Patil PK, Pillai S (2015) Effect of Vibrio bacterial product CIBASTIM administration on productivity in commercial tiger shrimp Penaeus monodon culture ponds in Gujarat. J Appl Aquac 27:107–112

    Article  Google Scholar 

  • Soto-Rodriguez SA, Gomez-Gil B, Lozano-Olvera R, Betancourt-Lozano M, Morales-Covarrubias MS (2015) Field and experimental evidence of Vibrio parahaemolyticus as the causative agent of acute hepatopancreatic necrosis disease of cultured shrimp (Litopenaeus vannamei) in Northwestern Mexico. Appl Environ Microbiol 81:1689–1699

    Article  PubMed  Google Scholar 

  • Spor A, Koren O, Ley R (2011) Unravelling the effects of the environment and host genotype on the gut microbiome. Nat Rev Microbiol 9:279–290

    CAS  Article  PubMed  Google Scholar 

  • Stecher B, Chaffron S, Käppeli R, Hapfelmeier S, Freedrich S, Weber TC, Kirundi J, Suar M, McCoy KD, von Mering C (2010) Like will to like: abundances of closely related species can predict susceptibility to intestinal colonization by pathogenic and commensal bacteria. PLoS Pathog 6:e1000711

    Article  PubMed  PubMed Central  Google Scholar 

  • Stephens WZ, Burns AR, Stagaman K, Wong S, Rawls JF, Guillemin K, Bohannan BJ (2016) The composition of the zebrafish intestinal microbial community varies across development. ISME J 10:644–654

    Article  Google Scholar 

  • Sullam KE, Essinger SD, Lozupone CA, O’Connor MP, Rosen GL, Knight R, Kilham SS, Russell JA (2012) Environmental and ecological factors that shape the gut bacterial communities of fish: a meta-analysis. Mol Ecol 21:3363–3378

    Article  PubMed  Google Scholar 

  • Summerfelt ST, Sharrer MJ, Tsukuda SM, Gearheart M (2009) Process requirements for achieving full-flow disinfection of recirculating water using ozonation and UV irradiation. Aquac Eng 40:17–27

    Article  Google Scholar 

  • Sun DL, Jiang X, Wu QL, Zhou NY (2013) Intragenomic heterogeneity of 16S rRNA genes causes overestimation of prokaryotic diversity. Appl Environ Microbiol 79:5962–5969

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Sung H, Hsu S, Chen C, Ting Y, Chao W (2001) Relationships between disease outbreak in cultured tiger shrimp (Penaeus monodon) and the composition of Vibrio communities in pond water and shrimp hepatopancreas during cultivation. Aquaculture 192:101–110

    Article  Google Scholar 

  • Tannock GW (2004) A special fondness for lactobacilli. Appl Environ Microbiol 70:3189–3194

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Thitamadee S, Prachumwat A, Srisala J, Jaroenlak P, Salachan PV, Sritunyalucksana K, Flegel TW, Itsathitphaisarn O (2016) Review of current disease threats for cultivated penaeid shrimp in Asia. Aquaculture 452:69–87

    Article  Google Scholar 

  • Tseng I, Chen JC (2004) The immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus under nitrite stress. Fish Shellfish Immunol 17:325–333

    CAS  Article  PubMed  Google Scholar 

  • Vanwonterghem I, Jensen PD, Dennis PG, Hugenholtz P, Rabaey K, Tyson GW (2014) Deterministic processes guide long-term synchronised population dynamics in replicate anaerobic digesters. ISME J 8:2015–2018

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Verschuere L, Rombaut G, Sorgeloos P, Verstraete W (2000) Probiotic bacteria as biological control agents in aquaculture. Microbiol Mol Biol Rev 64:655–671

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Vine NG, Leukes WD, Kaiser H (2006) Probiotics in marine larviculture. FEMS Microbiol Rev 30:404–427

    CAS  Article  PubMed  Google Scholar 

  • Vinod M, Shivu M, Umesha K, Rajeeva B, Krohne G, Karunasagar I, Karunasagar I (2006) Isolation of Vibrio harveyi bacteriophage with a potential for biocontrol of luminous vibriosis in hatchery environments. Aquaculture 255:117–124

    CAS  Article  Google Scholar 

  • Walker PJ, Mohan C (2009) Viral disease emergence in shrimp aquaculture: origins, impact and the effectiveness of health management strategies. Rev Aquac 1:125–154

    Article  Google Scholar 

  • Wang Y, Li J, Lin J (2008) Probiotics in aquaculture: challenges and outlook. Aquaculture 281:1–4

    Article  Google Scholar 

  • Wen C, Xue M, Liang H, Zhou S (2014) Evaluating the potential of marine Bacteriovorax sp. DA5 as a biocontrol agent against vibriosis in Litopenaeus vannamei larvae. Vet Microbiol 173:84–91

    Article  PubMed  Google Scholar 

  • Wu J, Xiong J, Hu C, Shi Y, Wang K, Zhang D (2015) Temperature sensitivity of soil bacterial community along contrasting warming gradient. Appl Soil Ecol 94:40–48

    Article  Google Scholar 

  • Xiong J, Zhu J, Wang K, Wang X, Ye X, Liu L, Zhao Q, Hou M, Qiuqian L, Zhang D (2014a) The temporal scaling of bacterioplankton composition: high turnover and predictability during shrimp cultivation. Microb Ecol 67:256–264

    Article  PubMed  Google Scholar 

  • Xiong J, Zhu J, Zhang D (2014b) The application of bacterial indicator phylotypes to predict shrimp health status. Appl Microbiol Biotechnol 98:8291–8299

    CAS  Article  PubMed  Google Scholar 

  • Xiong J, Chen H, Hu C, Ye X, Kong D, Zhang D (2015a) Evidence of bacterioplankton community adaptation in response to long-term mariculture disturbance. Sci Rep 5:15274

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Xiong J, Wang K, Wu J, Qiuqian L, Yang K, Qian Y, Zhang D (2015b) Changes in intestinal bacterial communities are closely associated with shrimp disease severity. Appl Microbiol Biotechnol 99(16):6911–6919

    CAS  Article  PubMed  Google Scholar 

  • Yan Q, Li J, Yu Y, Wang J, He Z, Van Nostrand JD, Kempher ML, Wu L, Wang Y, Liao L, Li X, Wu S, Ni J, Wang C, Zhou J (2016) Environmental filtering decreases with fish development for the assembly of gut microbiota. Environ Microbiol. doi:10.1111/1462-2920.13365

    Google Scholar 

  • Yang YW, Chen MK, Yang BY, Huang XJ, Zhang XR, He LQ, Zhang J, Hua ZC (2015) Use of 16S rRNA gene-targeted group-specific primers for real-time PCR analysis of predominant bacteria in mouse feces. Appl Environ Microbiol 81:6749–6756

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Zhang D, Wang X, Xiong J, Zhu J, Wang Y, Zhao Q, Chen H, Guo A, Wu J, Dai H (2014) Bacterioplankton assemblages as biological indicators of shrimp health status. Ecol Indic 38:218–224

    CAS  Article  Google Scholar 

  • Zhang M, Sun Y, Chen L, Cai C, Qiao F, Du Z, Li E (2016) Symbiotic bacteria in gills and guts of Chinese mitten crab (Eriocheir sinensis) differ from the free-living bacteria in water. PLoS One 11:e0148135

    Article  PubMed  PubMed Central  Google Scholar 

  • Zheng Y, Yu M, Liu Y, Su Y, Xu T, Yu M, Zhang X (2016) Comparison of cultivable bacterial communities associated with Pacific white shrimp (Litopenaeus vannamei) larvae at different health statuses and growth stages. Aquaculture 451:163–169

    Article  Google Scholar 

  • Zhou J, Fang W, Yang X, Zhou S, Hu L, Li X, Qi X, Su H, Xie L (2012) A nonluminescent and highly virulent Vibrio harveyi strain is associated with “bacterial white tail disease” of Litopenaeus vannamei shrimp. PLoS One 7:e29961

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was supported by the Zhejiang Province Public Welfare Technology Application Research Project (2016C32063), the Spark Program of China (2015GA701024), the Open Fund of Key Laboratory of Marine Genetic Resources, the Third Institute of Oceanography (HY201601), and the KC Wong Magna Fund of Ningbo University.

Author information

Affiliations

  1. School of Marine Sciences, Ningbo University, Ningbo, 315211, China

    Jinbo Xiong, Wenfang Dai & Chenghua Li

  2. Collaborative Innovation Center for Zhejiang Marine High-Efficiency and Healthy Aquaculture, Ningbo, 315211, China

    Jinbo Xiong, Wenfang Dai & Chenghua Li

Authors
  1. Jinbo Xiong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wenfang Dai
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chenghua Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jinbo Xiong.

Ethics declarations

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

The authors declare that they have no conflict of interest.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Xiong, J., Dai, W. & Li, C. Advances, challenges, and directions in shrimp disease control: the guidelines from an ecological perspective. Appl Microbiol Biotechnol 100, 6947–6954 (2016). https://doi.org/10.1007/s00253-016-7679-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-016-7679-1

Keywords

  • Shrimp disease
  • Ecological theory
  • Age-dependent therapeutic strategies
  • Indicative assemblages
  • Quantitative prediction