Hemolymph Microbiomes of Three Aquatic Invertebrates asRevealed by a New Cell Extraction Method

Xinxu Zhang,a,b Zaiqiao Sun,a,b Xusheng Zhang,a,b Ming Zhang,a,b Shengkang Lia,b

aGuangdong Provincial Key Laboratory of Marine Biotechnology, Shantou University, Shantou, ChinabMarine Biology Institute, Shantou University, Shantou, China

ABSTRACT Symbiotic microorganisms have been found in the hemolymph (blood)of many aquatic invertebrates, such as crabs, shrimp, and oysters. Hemolymph is acritical site in the host immune response. Currently, studies on hemolymph microor-ganisms are mostly performed with culture-dependent strategies using selective me-dia (e.g., thiosulfate-citrate-bile salts-sucrose [TCBS], 2216E, and LB) for enumeratingand isolating microbial cells. However, doubts remain about the “true” representa-tion of the microbial abundance and diversity of symbiotic microorganisms in hemo-lymph, particularly for uncultivable microorganisms, which are believed to be moreabundant than the cultured microorganisms. To explore this, we developed a culture-independent cell extraction method for separating microbial cells from the hemo-lymph of three aquatic invertebrates (Scylla paramamosain [mud crab], Litopenaeusvannamei [whiteleg shrimp], and Crassostrea angulata [Portuguese oysters]) involvingfiltration through a 5-�m-pore-size mesh filter membrane (the filtration method). Acombination of the filtration method with fluorescence microscopy and high-throughput sequencing technique provides insight into the abundances and diver-sity of the total microbiota in the hemolymph of these three invertebrates. Morethan 2.6 � 104 cells/ml of microbial cells dominated by Escherichia-Shigella and Halo-monas, Photobacterium and Escherichia-Shigella, and Pseudoalteromonas and Arcobacterwere detected in the hemolymph of Scylla paramamosain, Litopenaeus vannamei, andCrassostrea angulata, respectively. A parallel study for investigating the hemolymph mi-crobiomes by comparing the filtration method and a culture-dependent method (theplate count method) showed significantly higher microbial abundances (between 26-and 369-fold difference; P � 0.05) and less biased community structures of the filtra-tion method than those of the plate count method. Furthermore, hemolymph of thethree invertebrates harbored many potential pathogens, including Photobacterium,Arcobacter, and Vibrio species. Finally, the filtration method provides a solution thatimproves the understanding of the metabolic functions of uncultivable hemolymphmicroorganisms (e.g., metagenomics) devoid of host hemocyte contamination.

IMPORTANCE Microorganisms are found in the hemolymph of invertebrates, a criti-cal site in the host immune response. Currently, studies on hemolymph microorgan-isms are mostly performed with culture-dependent strategies. However, doubts re-main about the “true” representation of the hemolymph microbiome. This studydeveloped a culture-independent cell extraction method that could separate micro-bial cells from the hemolymph of three aquatic invertebrates (S. paramamosain, L.vannamei, and C. angulata) based on filtration through a 5-�m-pore-size mesh filtermembrane (the filtration method). A combination of the filtration method with fluo-rescence microscopy and a high-throughput sequencing technique provides insightinto the abundances and diversity of the total microbiota in the hemolymph ofthese three invertebrates. Our results demonstrate that the hemolymph of aquaticinvertebrates harbors a much higher microbial abundance and more distinct micro-bial community composition than previously estimated. Furthermore, this work pro-

Received 20 December 2017 Accepted 2February 2018

Accepted manuscript posted online 16February 2018

Citation Zhang X, Sun Z, Zhang X, Zhang M, LiS. 2018. Hemolymph microbiomes of threeaquatic invertebrates as revealed by a new cellextraction method. Appl Environ Microbiol84:e02824-17. https://doi.org/10.1128/AEM.02824-17.

Editor Shuang-Jiang Liu, Chinese Academy ofSciences

Copyright © 2018 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Shengkang Li,lisk@stu.edu.cn.

INVERTEBRATE MICROBIOLOGY

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vides a less biased solution for studying the metabolic functions of uncultivable he-molymph microbiota devoid of host hemocyte contamination.

KEYWORDS aquatic invertebrates, hemolymph, microbiome, cell extraction

Diverse symbiotic microorganisms are important inhabitants in various sites of theirhost’s body, including the intestinal tract, epidermis, and gill. Previous

microbiome-related studies have focused on the gut microbiota of vertebrates, such ashumans, mice, and some fish species, revealing their significant roles in host digestion,immunity, and health (1, 2). For example, some anaerobic microorganisms in theintestine (e.g., Faecalibacterium and Anaerostipes spp.) ferment undigested carbohy-drates to short-chain fatty acids (SCFAs; including butyrate, propionate, and acetate).These SCFAs serve as energy sources for the colonic epithelium, take part in appetitecontrol, and exhibit anti-inflammatory effects (3). In some fish species, gut bacteria maycontribute to the degradation of plant-derived nonstarch polysaccharides that areindigestible by the host (e.g., Ruminococcus and Clostridium) (4–6). For aquatic inver-tebrates with important economic values (e.g., crabs, shrimp, and oysters), relatedmicrobiome studies focused on the microbial diversity in the host’s gut or hemolymphunder different health statuses (7, 8) or environmental conditions (9, 10).

Hemolymph is a critical site in host immune response and is considered “sterile”without proliferating microorganisms in healthy animals (11). Since the report ofhemolymph bacteria in Callinectes sapidus (12, 13), growing evidence has demonstratedthe presence of viable microorganisms in the hemolymph of some aquatic inverte-brates (Table 1). As previously reported, the hemolymph microbial abundance inhealthy invertebrates is generally between nondetectable levels and 103 CFU/ml, withVibrio, Acinetobacter, and Aeromonas being the prevalent groups (Table 1). Recentstudies have revealed that temperature significantly altered the microbial communitystructures in the hemolymph of Crassostrea gigas (14). Furthermore, a C-type lectinMjHeCL and a prophenoloxidase were shown to maintain the homeostasis between thehost and its hemolymph microbiota by inhibiting the proliferation of these microor-ganisms in Marsupenaeus japonicus, respectively (15, 16). However, some indigenoushemolymph microorganisms were hypothesized to compete with incoming microor-ganisms (17), they may stimulate immune molecules and cells to generate hostimmunity (18), and in some cases, they could be potential pathogens (19). Hence, thereis a need for a systematic characterization of the hemolymph microbiomes to establishtheir abundances and diversity.

Currently, studies on hemolymph microorganisms are mostly performed withculture-dependent strategies using selective media (e.g., thiosulfate-citrate-bile salts-sucrose [TCBS], 2216E, and LB) for enumerating and isolating microbial cells. However,this approach may under- or overestimate the abundance and diversity of somehemolymph microbial groups, since each medium can culture a small subset of thetotal microbial community (20). For instance, while TCBS medium is frequently used tocharacterize the total microbial cell counts and/or community compositions in somehemolymph microbiome-related studies (Table 1), this medium is particularly suitablefor Vibrio spp., as it inhibits the growth of other microorganisms. This may results in afalse “sterile” hemolymph condition if Vibrio spp. are absent in the microbial community(14). In this regard, culture-independent strategies (including enumeration of microbialcells by fluorescence microscopy and characterization of microbial community struc-tures and metabolic functions by high-throughput sequencing of the 16S rRNA genesas well as metagenomics [21, 22]) are promising alternatives. Unfortunately, one majorproblem that hinders the application of culture-independent methods is the extremelylow abundance of microbial cells in hemolymph (�104 CFU/ml; Table 1) compared tothat of hemocytes (�106 hemocytes/ml) (see Fig. S1 in the supplemental material). Thecellular volume of a hemocyte is much larger than that of a microbial cell (Fig. 1). Dueto the abundance and size differences, some microbial cells could be covered byhemocytes when viewed under a fluorescence microscope. In addition, the amount of

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extracted microbial DNA is much less than that of hemocyte DNA, which would lead toan underestimation of the abundance of hemolymph microorganisms; therefore, onlylimited genomic information of these microorganisms would be obtained by high-throughput sequencing techniques.

In this study, we aimed to develop a culture-independent cell extraction method forseparating microbial cells from the hemolymph of three aquatic invertebrates, Scyllaparamamosain (mud crab), Litopenaeus vannamei (whiteleg shrimp), and Crassostreaangulata (Portuguese oysters). This new method would facilitate the extraction andseparation of hemolymph microbial cells, which would provide better insights into theabundances and diversity of hemolymph microorganisms of invertebrates.

RESULTSMorphological observation of hemocytes and microorganisms in the hemo-

lymph. When viewed under a microscope, most of the hemocytes in the hemolymphwere round or fusiform (Fig. 1a, d, and g) with diameters in the range of 7.5 to 15.3 �m,7.0 to 13.2 �m, and 6.1 to 11.9 �m for S. paramamosain, L. vannamei, and C. angulata,respectively. The morphologies of the observed hemocytes were similar to thosereported in previous studies, including hyalinocytes, semigranulocytes, and granulo-cytes in S. paramamosain (23) and L. vannamei (24), and hyalinocytes, granulocytes, andbrown cells in C. angulata (25). Microbial cells were rod or spherical shaped, with adiameter range of 0.5 to 2.0 �m. The microbial cells appeared as single free-living cellsthat were not clumped or otherwise associated with hemocytes (Fig. 1a, d, and g).Notably, the fluorescence intensity levels of the hemocytes as stained by SYBR GreenI were much higher than those of the microbial cells.

Permeability of the 5-�m-pore-size mesh filter membrane. We first tested thepermeability of the 5-�m-pore-size mesh membrane to hemocytes and microbial cellsin the hemolymph of the three species. For S. paramamosain, after filtering through the5-�m-pore-size mesh membrane, most of the hemocytes in the hemolymph wereretained on the membrane, while the microbial cells passed through the membrane(Fig. 1a and b). A similar observation was obtained for L. vannamei (Fig. 1d and e) and

TABLE 1 Microbial cell counts and community compositions in some hemolymph microbiome-related studies using culture-dependentmethods

Species Culture mediuma Microbial abundanceb Major microbial group(s) Reference or source

Scylla paramamosain 2216E 55 to 1,675 CFU/ml Vibrio spp., Tenacibaculum spp., Shewanella spp. This studyCallinectes sapidus TGY broth with

seawater1,876 MPN/ml V. parahaemolyticus Tubiash et al. (12), Sizemore

et al. (13)Limulus polyphemus 2216E ND to �100 MPN/ml NA Brandin and Pistole (29)

Litopenaeus vannamei 2216E 950 to 9,410 CFU/ml Vibrio spp., Acinetobacter spp., Shewanella spp. This studyTCBS 30 to 460 MPN/ml Vibrio spp. Albuquerque-Costa et al. (73)TCBS 200 to 3,000 CFU/ml Vibrio spp. Gomez-Gil et al. (74)

Marsupenaeus japonicus 2216E 10 to 1,000 CFU/ml Vibrio spp., Pseudoalteromonas spp. Wang et al. (15)NA �100 CFU/ml Vibrio spp., Escherichia spp. Kaizu et al. (75)LB, BHI Low Vibrio spp., Enterobacteriaceae Fagutao et al. (16)

Penaeus monodon LB with 2% NaCl �1,000 CFU/ml NA Ponprateep et al. (76)Procambarus clarkii NA NA Acinetobacter spp., Aeromonas spp., Arthrobacter

spp., Bacillus spp.Scott and Thune (77)

Homarus americanus BHI NA NA Cornick and Stewart (78)Crassostrea angulata 2216E 140 to 810 CFU/ml Vibrio spp., Tenacibaculum spp., Ruegeria spp. This study

Crassostrea gigas Marine broth 2216,blood agar

140 to 560 CFU/ml Pseudomonas spp., Alteromonas spp., Vibriospp., Aeromonas spp.

Olafsen et al. (30)

TCBS ND to 202 CFU/ml Epsilonproteobacteria, Gammaproteobacteria,Flavobacteriac

Lokmer and Mathias Wegner (14)

Modiolus modiolus Marine broth 2216,blood agar

26,000 CFU/ml Pseudomonas spp., Vibrio spp., Aeromonas spp. Olafsen et al. (30)

Anodonta cygnea Plate count agar 150 CFU/ml Vibrio metschnikovii, Aeromonas sobria Antunes et al. (79)aTGY, tryptone-glucose-yeast; TCBS, thiosulfate-citrate-bile salts-sucrose; NA, not applicable; LB, Luria-Bertani broth; BHI, brain heart infusion broth.bND, not detected; MPN, most probable number.cThese groups were determined by a culture-independent method.

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C. angulata (Fig. 1g and h). However, few hemocytes were able to escape through thepores of the 5-�m-pore-size mesh membrane for each species, with an averagefrequency of 1 to 2 hemocytes in 20 fields of views (Fig. 1c, f, and i).

To test the effect of the filtration step using the 5-�m-pore-size mesh membrane onmicrobial cell recovery or potential losses, one Vibrio parahaemolyticus bacterial strainwas introduced as a simulant of hemolymph microorganisms, and its initial number ofcells was set at 2.2 � 104, as determined by fluorescence microscopy (Fig. 2a). A slightlylower abundance of the V. parahaemolyticus cells occurred when they passed throughthe 5-�m-pore-size mesh membrane (V-5 �m; 1.9 � 104 cells; P � 0.05). Furthermore,in order to simulate the hemolymph condition in situ, hemocytes of S. paramamosainwere added to the V. parahaemolyticus cells at a final number of �2.0 � 106 hemocytes(Fig. S1). After filtration through a 5-�m-pore-size mesh membrane, the microbial countof the V. parahaemolyticus-hemocyte mixture (V-H-5 �m) was 1.8 � 104 cells, which wasnot statistically different from the V-5 �m count (P � 0.05; Fig. 2a). Similar results wereobserved in groups with an initial V. parahaemolyticus count of 3.2 � 106 cells (Fig. 2b).In total, the recovery ratio of V. parahaemolyticus cells after filtration through the5-�m-pore-size mesh membrane was between 82% and 87%, although significantly

FIG 1 Fluorescence microscopy showing hemocytes and microbial cells in the hemolymph of S. paramamosain (a to c), L.vannamei (d to f), and C. angulata (g to i). The blue arrows indicate hemocytes, and the red arrows indicate microbial cells. (a,d, and g) Untreated hemolymph samples. (b, c, e, f, h, and i) Hemolymph samples which have been passed through a5-�m-pore-size mesh membrane. The scale bars are 10 �m.

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lower number of microbial cells was observed in the group V-H-5 �m than in the groupV (V. parahaemolyticus cells were counted on a 0.2-�m-pore-size mesh membrane) (P �

0.05).However, the permeability of the 2-�m-pore-size mesh membrane to microbial cells

was poor, and only between 2% and 35% of the total V. parahaemolyticus cellsremained after filtration (Fig. S2a and b). For the 8-�m-pore-size mesh membrane,many hemocytes could pass through its pores, thus hindering the detection andenumeration of the V. parahaemolyticus cells (Fig. S3), although it showed goodpermeability to these microbial cells (Fig. S2c and d).

Hemolymph microbial abundance. In each species (S. paramamosain, L. vannamei,and C. angulata), significant variations in hemolymph microbial abundance wereobserved between the filtration method and the plate count method (Fig. 3). Theabundances of hemolymph microorganisms obtained by the filtration method werebetween 26- and 369-fold higher than those by the plate count method (Table S1; P �

0.05). The coefficient of variation (CV) of the filtration method was also lower than thatof the plate count method (Table S1). For example, for the same 6 S. paramamosaincrabs, the average hemolymph microbial abundance as determined by the filtrationmethod was 2.6 � 104 cells/ml, with a CV of 0.16, while the average hemolymphmicrobial abundance as determined by the plate count method was 6.6 � 102 CFU/ml,with a CV of 0.92 (Fig. 3 and Table S1). In the case of L. vannamei, the averagehemolymph microbial abundance was 1.3 � 105 cells/ml (CV � 0.11, filtration method)versus 5.1 � 103 CFU/ml (CV � 0.66, plate count method). Similarly, for C. angulata, theaverage hemolymph microbial abundance was 1.7 � 105 cells/ml (CV � 0.07, filtrationmethod) or 4.5 � 102 CFU/ml (CV � 0.58, plate count method).

The hemolymph microbial abundance of 6 moribund S. paramamosain crabs suf-fering from hydropenia was further analyzed using the two methods, with the resultsshowing a significantly higher abundance of hemolymph microorganisms than those inhealthy individuals (Fig. 4a). The average hemolymph microbial abundances as deter-mined by the filtration method and the plate count method were 3.6 � 106 cells/mland 3.9 � 103 CFU/ml, respectively. Notably, the increased microbial abundance inmoribund crabs was more than 100-fold higher than that in healthy individuals as

FIG 2 Assessment of permeability of the 5-�m-pore-size mesh filter membrane. Numbers of the initial V. parahaemolyticus cells wereset at 2.2 � 104 cells (a) and 3.2 � 106 cells (b). V, V. parahaemolyticus cells were counted on a 0.2-�m-pore-size mesh membrane.V-5 �m, V. parahaemolyticus cells were filtered through a 5-�m-pore-size mesh membrane and counted on a 0.2-�m-pore-size meshmembrane. V-H-5 �m, V. parahaemolyticus cells were mixed with hemocytes of S. paramamosain, filtered through a 5-�m-pore-sizemesh membrane, and counted on a 0.2-�m-pore-size mesh membrane. Significant differences (P � 0.05) are represented by differentletters.

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determined by the filtration method (2.6 � 104 cells/ml; Fig. 3), while it was only 6-foldhigher than that in healthy crabs as determined by the plate count method (6.6 � 102

CFU/ml). To compare the differences in the cell enumeration between the two meth-ods, an equal amount of pure V. parahaemolyticus bacteria (isolated from hemolymphof a diseased S. paramamosain [26]) was enumerated. The result showed that theaverage microbial abundance as determined by the filtration method was 3.7 � 109

FIG 3 Microbial abundances in the hemolymph of the three invertebrates as determined by the filtrationmethod and the plate count method. The units CFU/ml and cells/ml are used for the plate count methodand the filtration method, respectively. Each dot indicates an individual sample. The horizontal barsrepresent the medians. The asterisks indicate a significant difference (P � 0.05).

FIG 4 Microbial abundance in the hemolymph of moribund S. paramamosain (a) and of pure V. parahaemolyticus bacteria (b) as determined by the filtrationmethod and the plate count method. The units CFU/ml and cells/ml are used for the plate count method and the filtration method, respectively. Each dotindicates an individual sample. The horizontal bars represent the medians. The asterisks indicate a significant difference (P � 0.05).

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cells/ml, which was about 4-fold higher than that by the plate count method (9.2 � 108

CFU/ml; Fig. 4b).Hemolymph microbial community composition. In general, distinct hemolymph

microbial community compositions were observed between the two methods in thisstudy (Fig. 5). The nonmetric multidimensional scaling (NMDS) plot showed clearseparation of microbial communities by the filtration method and the plate countmethod for each species, and with the triplicate individuals in each group clusteringtogether (except for the S. paramamosain-plate count method [Sp-P] group; Fig. 6). Thedifferences in the relative abundances of hemolymph microorganisms between the twomethods were further compared by MetaStat analysis (27). In S. paramamosain, thehemolymph microbial community obtained by the filtration method and tested byMetaStat analysis was dominated by Escherichia-Shigella and Halomonas (Table S2; P �

0.05), with relative abundances of 37.8 to 42.3% and 11.7 to 39.1%, respectively.However, Tenacibaculum (10.3 to 82.6%), Vibrio (0.006 to 45.7%) and Shewanella (0.01to 40.4%) spp. were more frequently detected by the plate count method. Otherbacterial groups with significant differences in the relative abundance between the two

FIG 5 Hemolymph bacterial community compositions and hierarchical clustering of the three invertebrates based on the 16SrRNA gene sequence abundance. Color bars indicate the percentage of sequences in each designated genus, with asterisksindicating OTUs that could not be assigned to genus level in the Greengenes 16S rRNA gene database. Only the top 28abundances of bacterial lineages are shown, and the rest are grouped into “Others.” Hierarchical cluster dendrogram ofmicrobial communities are based on Bray-Curtis distance matrix. The scale bar indicates distance in length. Sp, S. paramamo-sain; Lv, L. vannamei; Ca, C. angulata; F, the filtration method; P, the plate count method. Three samples were sequenced foreach group.

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methods included Photobacterium, Pelagibacterium, and Comamonadaceae (Table S2).In the case of L. vannamei, the hemolymph microbial community obtained by thefiltration method was dominated by Photobacterium (64.6 to 82.0% in relative abun-dance), Escherichia-Shigella (4.5 to 8.9%), and Halomonas (1.9 to 8.4%) spp. (Fig. 5 andTable S3; P � 0.05). However, the predominant microorganisms grown on the 2216Eagar plate were Vibrio (38.7 to 56.4%), Acinetobacter (25.0 to 37.2%), and Shewanella (2.0to 8.7%) spp. (P � 0.05). Other bacterial groups with significant differences in relativeabundance between the two methods included Tenacibaculum, Pseudomonas, andBacillus spp. (Table S3). In C. angulata, Pseudoalteromonas (46.2 to 71.9%) and Arcobac-ter (13.0 to 24.0%) spp. dominated the microbial community when the filtration methodwas used (Fig. 5 and Table S4; P � 0.05), while higher abundances of Ruegeria (9.8 to41.3%), Tenacibaculum (2.2 to 42.5%), and Vibrio (9.7 to 45.9%) spp. were detected byuse of the plate count method (P � 0.05). Other bacterial groups with significantdifferences in relative abundance between the two methods included Shewanella spp.,Pelagibacterium spp., Labrenzia spp., and Alteromonadaceae (Table S4).

In total, alpha-diversity indices (expressed as phylogenetic diversity [PD] whole treeand Chao 1) in each analyzed invertebrate species were higher in communities deter-mined by the filtration method than by the plate count method (Fig. 7). Among thethree invertebrates, the most discriminating bacterial groups (calculated from lineardiscriminant analysis [LDA] scores at the genus level) as determined by the filtrationmethod were Escherichia-Shigella and Halomonas from S. paramamosain, Photobacte-rium and Hydrogenophaga from L. vannamei, and Arcobacter from C. angulata (Fig. S4).Notably, a cluster analysis based on Bray-Curtis distance showed that microbial com-munities obtained by the plate count method formed a single lineage, although theyare from the hemolymph of three different invertebrates (Fig. 5).

DISCUSSION

Direct application of culture-independent methods (such as observation of hemo-lymph microorganisms under the microscope) is extremely difficult, because (i) the

FIG 6 An NMDS plot showing variation in the hemolymph microbial community structures of the threeinvertebrates as determined by the filtration method and the plate count method. The distances weredetermined using the Bray-Curtis method with relative abundances of microorganisms at the genus level.Sp, S. paramamosain; Lv, L. vannamei; Ca, C. angulata; F, the filtration method; P, the plate count method.Three samples were sequenced for each group.

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abundance of hemocytes (�106 hemocytes/ml; Fig. S1) is much higher than that ofmicrobial cells (�105 cells/ml; Fig. 3), (ii) the “small” microbial cells could be covered by“big” hemocytes, thus hindering the detection of the microbial cells (Fig. 1), and (iii) thefluorescence intensity of hemocytes is much higher than that of microbial cells (Fig. 1and S3). In this study, we developed a culture-independent method that could effi-ciently extract microbial cells from the hemolymph of three aquatic invertebrates byusing a 5-�m-pore-size mesh filter membrane (Fig. 8), thereby revealing much higherabundance and diversity of hemolymph microbiota than previously estimated (Fig. 3 to5 and 7).

Aquatic invertebrates harbor more hemolymph microorganisms than previ-ously estimated. In each of S. paramamosain, L. vannamei, and C. angulata, thehemolymph microbial abundance as determined by the filtration method was muchhigher than that using the plate count method (more than 26-fold difference; Fig. 3),although there was approximately a 4-fold inherent difference between these two cellenumeration methods (as assessed using an equal amount of a pure bacterial strain;Fig. 4b). The inherent difference between the two methods is possibly due to the failurein culturing some dormant and/or inactive cells by the plate count method, while thesecells could still be detected by the filtration method if the cell nucleus maintains its

FIG 7 Violin plots showing distribution of alpha diversity indices of the three invertebrates as determined by the filtration method and the platecount method. (a) PD whole-tree index. (b) Chao 1 index. The white dots indicate medians. The box covers between the 25% and 75% quantiles.Significance was assessed using the Kruskal-Wallis test. Sp, S. paramamosain; Lv, L. vannamei; Ca, C. angulata; F, the filtration method; P, the platecount method.

FIG 8 A flow diagram showing the filtration method, its further applications, and the plate count method.

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integrity. Moreover, great differences in the hemolymph microbial abundance ofmoribund S. paramamosain cells were observed between the two methods (3.6 � 106

cells/ml by the filtration method versus 3.9 � 103 CFU/ml by the plate count method;Fig. 4a). This observation indicates that hydropenia might have caused a decrease inthe immunity of the crabs and therefore an unbalanced homeostasis occurred betweenthe host and its hemolymph microbiota (28). The increased microorganisms in thesemoribund crabs were likely endosymbiotic and uncultivable, since no apparent externalinjury was applied or detected (e.g., injection or wounds). Through application of thefiltration method in this study, almost all of the hemocytes in the hemolymph wereremoved by the 5-�m-pore-size mesh membrane, and an estimated 82 to 87% ofthe total microbial cells were able to pass through the 5-�m-pore-size mesh membrane(Fig. 2). The filtration step was also carefully modified with different pore sizes of thefilter membrane (2 �m, 5 �m, and 8 �m), resulting in the best-optimized conditions ofthe 5-�m-pore-size mesh membrane (i.e., higher removal ratio of hemocytes and lowerloss ratio of microorganisms; Fig. 2, S2, and S3).

The 2216E medium is a commonly used organic-rich medium (containing 5 g/litertryptone and 1 g/liter yeast extract) for counting and characterizing the microbialcommunities in the hemolymph of some aquatic invertebrates, including M. japonicus(15), Limulus polyphemus (29), Modiolus modiolus, and C. gigas (30). The significantlylower cell counts as determined by the plate count method are possibly because somemembers of the hemolymph microorganisms failed to grow on the 2216E medium. Thiscould be probably caused by unfavorable energy or nutrient supplementation (20), theabsence of certain growth signals (31), insufficient incubation time (32), inhibition bybacteriocin-producing microorganisms (33), or dependence on other microorganisms(34). Moreover, some slow-growing or oligotrophic microorganisms in the hemolymphmay form minicolonies which could not be observed with the naked eye on the agarplate (35). In this case, a combination of the filtration method and fluorescencemicroscopy improved observation (Fig. 1) and enumeration accuracy (CV � 0.16; TableS1) of the microbial cells without cultivation, therefore providing a true reflection of thetotal hemolymph microbial community in situ (e.g., microbial cell sizes and shapes andtheir association with hemocytes). Our results showed that previous studies on theabundances of microorganisms in the hemolymph of healthy and/or diseased inverte-brates were probably underestimated, as they are mostly performed based on culture-dependent methods, such as the plate count method (Table 1).

Distinct hemolymph microbial community composition as determined by thetwo methods. Although Vibrio spp. were frequently detected as the predominantgroup in the hemolymph of various healthy aquatic invertebrates as determined byculture-dependent methods, such as the plate count method (Table 1 and Fig. 5), therelative abundance of Vibrio spp. never reached 7% in the three invertebrates asdetermined by the filtration method in this study (Fig. 5). Given that many Vibrio spp.may grow rapidly on 2216E agar plates, they may further inhibit the growth of othermicroorganisms by competing for nutrients or space (36). Therefore, our results suggestthat Vibrio spp. probably represented a “pseudo”-major bacterial group in the hemo-lymph of these healthy invertebrates as determined using the culture-dependentmethods (Table 1). In total, the major hemolymph microbial groups detected by thefiltration method (i.e., Escherichia-Shigella and Halomonas in S. paramamosain, Photo-bacterium and Escherichia-Shigella in L. vannamei, and Pseudoalteromonas and Arcobac-ter in C. angulata) were either uncultivable or inhibited by other cultivable microor-ganisms (e.g., Vibrio, Tenacibaculum and/or Shewanella spp.) on the 2216E medium,thus leading to a biased description of the total hemolymph microbiota by the platecount method. This may also explain the reason why similar microbial communitystructures were detected by use of the plate count method (based on Bray-Curtisdistance), although they were from three different invertebrate species (Fig. 5).

A few bacterial groups were solely detected by the plate count method, includingBacillus, Tenacibaculum, Glutamicibacter, and/or Morganella spp. in L. vannamei and/orC. angulata (Fig. 5). This is possibly because these bacteria tightly adhered to the

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hemocytes or even located in the hemocytes, and they were retained on the 5-�m-pore-size mesh membrane as determined by the filtration method. It has been reportedin crabs and shrimp that hemocyte phagocytosis and agglutination are important hostimmune defense mechanisms against potential pathogens and exogenous bacteria (37,38), and in some cases, they were not always associated with bacterial virulence (39).Moreover, some bacteria species may facilitate the phagocytosis and agglutinationprocesses through specific structures, such as the outer membrane protein OmpU (40),or they could produce extracellular substances, such as proteases, phospholipases,hemolysins, or cytotoxins (30, 41). This suggests that bacterial phagocytosis and/oragglutination with the hemocytes may occur spontaneously in the hemolymph of ahealthy animal, and this phenomenon was further confirmed by the phagocytosisobserved in S. paramamosain (Fig. S5). Unfortunately, we were unable to separate thesebacterial cells (e.g., Bacillus and Tenacibaculum spp.) from hemocytes using the filtrationmethod in this study. Other probable reasons that might have hindered the detectionof these bacterial groups included that: (i) some bacterial cells in the hemolymph weretoo big to pass through the 5-�m-pore-size mesh membrane (e.g., strains from Te-nacibaculum are rod-shaped, with lengths up to 30 �m [42]), or (ii) some microorgan-isms might be in spore form due to unfavorable environmental conditions (e.g., strainsfrom Bacillus); these spores may be resistant to the DNA extraction procedures used inthis study, and their intracellular 16S rRNA genes were not eventually released orsequenced (43).

Potential roles of the hemolymph microbiome. In the hemolymph of the three

aquatic invertebrates, many bacterial groups were probably marine-derived aerobic orfacultative anaerobic heterotrophs (Fig. 5), with hemolymph oxygen, carbohydrates(e.g., glucose), lipids, and amino acids as their potential energy and nutrient sources (44,45). Notably, some predominant microorganisms were potential pathogens as deter-mined by the filtration method and/or the plate count method. For instance, someVibrio spp. are known pathogens to many marine animals, including crabs (26), shrimp(46), and oysters (47), which have caused great economic losses in the aquacultureindustry (48). In the case of S. paramamosain, some Acinetobacter spp. are opportunisticpathogens in humans and some aquaculture animals (49). Similarly, in L. vannamei,some Photobacterium spp. are reported to infect fish through their skin and may causefish septicemia (50, 51); some Aeromonas spp. and Flavobacterium spp. are also fre-quently isolated from diseased fish and shrimp, causing hemorrhagic septicemia, finrot, or furunculosis (52, 53). For C. angulata, the detected Arcobacter and Tenacibaculumspp. are known pathogens that are reported to have caused considerable economiclosses in marine fish and mollusc aquaculture (54–56). Moreover, the hemolymph of thethree invertebrates also harbors some potential probiotics, which may protect the hostagainst pathogen infection through the production of antimicrobials or antibiotics (17).For example, it has been reported that the abundant Pseudoalteromonas spp. detectedin the hemolymph of C. angulata produce antimicrobials against pathogenic bacteria,such as Vibrio spp. (57, 58), and dietary administration of a Halomonas strain couldprotect the integrity of the intestinal mucosal layer in Fenneropenaeus chinensis (59).However, rapid proliferation of these microorganisms in the hemolymph may lead tohost diseases, as observed in this study of the moribund S. paramamosain organism(Fig. 4a) and in previous reports of M. japonicus (15, 16).

It has been reported that hemocytes could express basal levels of two antimicrobialpeptides (i.e., defensins and proline-rich peptides) in nondiseased Pacific oyster (C.gigas), suggesting that indigenous hemolymph microorganisms can stimulate immunemolecules and cells to generate host immunity (18). Meanwhile, these microorganismsresist the host immune system through various ways, such as the secretion of proteaseand degradation of antimicrobial peptides (60), reducing the affinity for antimicrobialpeptides by modifying the net anionic charge of their cell envelope (61) or even byimpairing phagosome maturation (62). Unfortunately, it was not possible to determine

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the exact functions of these hemolymph microorganisms due to the experimentallimitations of this study.

Conclusion. In this study, a culture-independent cell extraction method was devel-oped that could separate microbial cells from the hemolymph of three aquatic inver-tebrates (i.e., S. paramamosain, L. vannamei, and C. angulata) based on filtrationthrough a 5-�m-pore-size mesh membrane (the filtration method). Our results dem-onstrate that the hemolymph of aquatic invertebrates harbors a much higher microbialabundance and distinct microbial community composition than were previously esti-mated. Furthermore, this work provides a less biased solution for studying the meta-bolic functions of uncultivable hemolymph microbiota devoid of host hemocyte con-tamination. Our ongoing studies exploring the potential functions of the totalhemolymph microbiota using microbial metagenomics and metatranscriptomics in S.paramamosain, as well as others focusing on the host-microbiome homeostatic immu-nity in M. japonicus and Drosophila melanogaster, may help elucidate and give betterinsights into understanding the significant roles of hemolymph microbiome in a host’simmunity and health.

MATERIALS AND METHODSSample collection. All of the animals used in this study were healthy, with no signs of disease, and

were obtained from a local aquaculture farm in Shantou, Guangdong, China. The average weights of S.paramamosain, L. vannamei, and C. angulata animals were 108.1 g, 7.5 g, and 39.7 g, respectively.Because the three invertebrates have different requirements for growth and production, they areseparately cultured in three different facilities and fed different foods. Moribund S. paramamosain crabssuffering from hydropenia were prepared by incubating healthy crabs at 37°C for �12 h withoutseawater and were used for experimentation when they breathe but without movement. Hemolymphwas collected aseptically (disinfected with 75% [vol/vol] ethanol) from the arthropodial membrane at thebase of the walking legs of S. paramamosain, from the ventral sinus of L. vannamei, and from theadductor muscle of C. angulata, with a sterile syringe and 26-gauge needle into an equal volume ofprecooled sterile anticoagulant buffer (450 mM NaCl, 100 mM glucose, 26 mM citric acid, 30 mM sodiumcitrate [pH 4.6]). Due to the small volume of hemolymph from L. vannamei (�0.15 ml), the hemolymphsamples of 15 shrimp were pooled as one sample.

The animals were processed according to the “Regulations for the administration of affairs concern-ing experimental animals” established by Guangdong Provincial Department of Science and Technologyon the Use and Care of Animals (6). The experiments were approved by the Institutional Animal Care andUse Committee of Shantou University.

Assessment of permeability of the 5-�m-pore-size mesh filter membrane. A pure bacterialculture Vibrio parahaemolyticus was dispensed into a formalin solution (20 g/liter NaCl, 30 ml/literformalin) and diluted to �2 � 104 cells/ml (equivalent of the hemolymph microbial abundance in ahealthy crab) and �3 � 106 cells/ml (equivalent of the hemolymph microbial abundance in a moribundcrab), respectively. Three treatment approaches were used to assess the permeability of the 5-�m-pore-size mesh membrane (TMTP02500; Millipore): (i) 1 ml of the pure culture was directly passed through asterile filter (SX0002500; Millipore) with a 0.2-�m-pore-size mesh membrane (GTBP02500; Millipore; hereV), which indicated the total number of V. parahaemolyticus cells; (ii) 1 ml of the pure culture was filteredthrough a 5-�m-pore-size mesh membrane, followed by a 0.2-�m-pore-size mesh membrane (here, V-5�m), which indicated the remaining V. parahaemolyticus cells after filtration through the 5-�m-pore-sizemesh membrane; (iii) to simulate the hemolymph condition in situ, 1 ml of the pure culture was mixedwith an equal volume of hemocytes (�2 � 106 hemocytes), and then the mixture was filtered througha 5-�m-pore-size mesh membrane, followed by a 0.2-�m-pore-size mesh membrane (here V-H-5 �m),which indicated the remaining V. parahaemolyticus cells after filtration through the 5-�m-pore-size meshmembrane. The hemocytes were prepared by filtering the hemolymph of S. paramamosain with a5-�m-pore-size mesh membrane, and the membrane was then transferred to a 2-ml centrifuge tube. Onemilliliter of formalin solution was added to the tube and vortexed for 5 min (Vortex-Genie 2; Mo BioLaboratories, USA) and then the membrane was removed. The 0.2-�m-pore-size mesh membrane wasthen stained with SYBR Green I solution (1:40 [vol/vol] SYBR Green I in 1� Tris-EDTA buffer) for 20 min.The stain solution was removed, the membrane was placed onto a glass slide, and 25 �l of 10% (vol/vol)glycerine was added as an antifade agent. Cells were counted at �1,000 magnification using afluorescence microscope (Axioplan 2 Imaging; Zeiss, Germany) with a blue filter set. Each sample wasextracted and counted in triplicate. An average of 200 fields of view were counted for each membrane.The area of each field of view was set at 10,000 �m2, and the detection limit was �103 cells/ml for a 95%probability of detecting at least 1 cell, as described previously (21, 22). The blank control was performedwith autoclaved and 0.2-�m-pore-size mesh membrane-filtered Milli-Q water (18.2 M�; Millipore, USA)and processed with the same filtration and staining steps as the bacterial samples. In addition to the5-�m-pore-size mesh membrane, the permeabilities of the 2-�m-pore-size mesh membrane (SLAP02550;Millipore) and the 8-�m-pore-size mesh membrane (TETP02500; Millipore) were also assessed as de-scribed above.

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Enumeration of microbial cells and hemocytes in hemolymph. (i) The filtration method. Thehemolymph-anticoagulant mixture was passed through a sterile filter (SX0002500; Millipore) with a5-�m-pore-size mesh membrane, and the filtrate was collected in a new centrifuge tube. This stepremoved most of the hemocytes in the hemolymph and kept the microbial cells in the filtrate (Fig. 1).The microbial cells in the filtrate were fixed with an equal volume of sterile formalin solution (20 g/literNaCl, 30 ml/liter formalin) for 30 min. The mixture was filtered through a sterile filter (SX0002500;Millipore) with a 0.2-�m-pore-size mesh membrane. Microbial cells on the 0.2-�m-pore-size meshmembrane were stained and counted as described in the preceding paragraph (Fig. 8).

(ii) The plate count method. The hemolymph was collected and processed as described above. Onehundred microliters of the hemolymph-anticoagulant mixture was spread on a modified 2216E agarplate (20 g/liter NaCl, 5 g/liter tryptone, 1 g/liter yeast extract, 0.1 g/liter FeCl3, 15 g/liter agar [pH 7.6]).After incubation at 30°C for 36 h, the colonies were counted on each plate (Fig. 8). Triplicate plates wereperformed for each sample. The negative control was performed with autoclaved and 0.2-�m-pore-sizemesh membrane-filtered Milli-Q water and processed with the same inoculation and incubation steps asfor the hemolymph samples.

Enumeration of hemocytes in the hemolymph was performed using the 5-�m-pore-size meshmembrane and then stained and counted as described in the preceding paragraph.

DNA extraction, PCR amplification, and sequencing. For the filtration method, the filtrate col-lected after passing through a 5-�m-pore-size mesh membrane was further filtered through a 0.2-�m-pore-size mesh membrane (GTBP02500; Millipore) to collect the microbial cells. The 0.2-�m-pore-sizemesh membrane was then cut into �0.2-cm2 pieces with a flame-sterilized scissors and added to a drybead tube (PowerFecal DNA isolation kit; Mo Bio Laboratories, USA). For the plate count method, all thecolonies in each plate were collected with a flame-sterilized glass spreading rod and added to a dry beadtube. Total DNA was extracted using the PowerFecal DNA isolation kit (Mo Bio Laboratories), accordingto the manufacturer’s protocol.

The hypervariable V3–V4 region of the bacterial 16S rRNA genes was amplified by PCR using theprimers 341F (5=-XXXXXXXX-CCTAYGGGRBGCASCAG-3=) and 806R (5=-GGACTACNNGGGTATCTAAT-3=)(63, 64), where X represents the barcode sequence unique to each sample. PCRs were performed intriplicate 20-�l mixtures containing 4 �l of 5� FastPfu buffer, 2 �l of 2.5 mM dinucleoside triphosphates(dNTPs), 0.8 �l of each primer (5 �M), 0.4 �l of FastPfu DNA polymerase (TransStart, China), and 10 ngof DNA template. The PCR cycling conditions were as follows: 95°C for 5 min, followed by 27 cycles at95°C for 30 s, 55°C for 30 s, and 72°C for 45 s, and a final extension at 72°C for 5 min. The PCR productswere electrophoresed on 2% agarose gels, extracted and purified using the AxyPrep DNA gel extractionkit (Axygen, USA), according to the manufacturer’s instructions, and then quantified using aQuantiFluor-ST fluorometer (Promega, USA). The DNA blank extraction was performed without a sampleand processed with the same DNA extraction and PCR amplification kits as with the hemolymph samples.DNA extraction and PCR amplification were considered free of contamination if no target PCR band ofapproximately 500 bp was seen on an agarose gel for the blank DNA extraction and the PCR-negativecontrol. Equimolar concentrations of each purified PCR product were pooled and sequenced on anIllumina HiSeq 2500 platform (2 � 250 bp; Illumina, USA), according to standard protocols. Processing ofraw sequence data was performed as described elsewhere (21). Briefly, raw reads which contained a50-bp continuous fragment with an average quality score less than 30 and/or any ambiguities wereremoved. Filtered reads were merged together using FLASH (version 1.2.7 [65]). Merged sequences wereremoved if they contained more than six identical bases occurring continuously and/or any ambiguitiesor if the sequence length was �200 bp. Clean sequences were demultiplexed using the QIIME softwarepipeline (version 1.9.0 [66]) with a mapping file containing the sample identification (ID), barcode, andprimer sequence.

Microbial community composition and statistical analysis. The effective sequences used in thisstudy were obtained by removing the chimeric sequences, which were detected using the Gold databaseby UCHIME program (version 4.2.40 [67]). In order to compare the samples at the same sequencingdepth, 31,066 sequences were obtained for each sample by random sampling (Table S5). The sequenceswere then clustered into operational taxonomic units (OTUs) at 97% sequence similarity cutoff using theUPARSE package (version 7.0.1001 [68]). Each OTU was taxonomically assigned to the Greengenesdatabase (Second Genome) using assign_taxonomy.py in QIIME. Alpha-diversity indices (phylogeneticdiversity [PD] whole tree [69], and Chao 1 [70]) were calculated by alpha_diversity.py in QIIME. Ahierarchical cluster dendrogram of microbial communities was developed using upgma_cluster.py withan unweighted pair group method using average linkages (UPGMA) method. The NMDS analysis wasperformed in the R software (version3.2.0 [71]) based on the Bray-Curtis method with relative abun-dances of microorganisms at the genus level. The LDA effect size (LEfSe) analysis was performed with anLDA score threshold of �4.0, as described elsewhere (72).

The statistical significance of cell enumeration data between two groups was analyzed by SPSS 22.0software using the Mann-Whitney U test. The significance of alpha-diversity indices was assessed in Rusing the Kruskal-Wallis test. MetaStat analysis with Fisher’s exact test was performed in R to determinethe differences in the relative abundances of hemolymph microorganisms between the two methods(27).

Bacterial phagocytosis assay. A strain of V. parahaemolyticus was labeled with fluorescein isothio-cyanate (FITC) stain, as described previously (37). In brief, V. parahaemolyticus was washed twice with 1�phosphate-buffered saline (PBS), heated at 70°C for 30 min, and washed again with 0.1 M NaHCO3.Following incubation in 0.1 M NaHCO3 containing 0.1 mg/ml FITC for 1 h at room temperature, thebacteria were rinsed with 1� PBS until no FITC was visible in the wash. Each crab was injected via the

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arthropodial membrane at the bases of the walking legs with 100 �l FITC-labeled bacteria (1 � 109

cells/ml). Immediately (0 h) and 1 h after bacterial injection, the hemolymph was collected in a 5-mlsyringe containing 1 ml of anticoagulant buffer. The hemocytes were collected by centrifuging thehemolymph-anticoagulant mixture at 700 � g for 5 min at 4°C and then resuspended in 1 mlanticoagulant buffer containing 3% formalin. After incubation for 10 min and washing with 1� PBS twice,the fixed hemocytes were spread onto a poly-L-lysine-coated glass slide and incubated for 1 h. The slidewas further stained with 4=,6-diamidino-2-phenylindole (DAPI) for 10 min. After three washes with 1�PBS, the cells were observed for phagocytosis using a fluorescence microscope (Axio Imager M2; Zeiss,Germany). The negative-control crab was injected with 100 �l of 1� PBS and processed with the samecollection and staining steps as for the hemolymph samples.

Accession number(s). All the sequence data obtained in this study have been deposited in theNational Center for Biotechnology Information (NCBI) Sequence Read Archive under the accessionnumber SRP125819.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/AEM.02824-17.

SUPPLEMENTAL FILE 1, PDF file, 0.8 MB.

ACKNOWLEDGMENTSWe thank Edmond Sanganyado and Jude Juventus Aweya for critical discussions

and proofreading of the manuscript.This work was supported by grants from the National Natural Science Foundation of

China (grant 41706185), Guangdong Provincial Project of Science and Technology(grant 2017B020204003), and the China Postdoctoral Science Foundation (grant2016M602501).

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