Brugia malayi L3 to L4 development
1
Insights into the L3 to L4 developmental program through proteomics 1
2
Sasisekhar Bennurua, Zhaojing Mengb, James McKerrowc, Sara Lustigmand and 3
Thomas B Nutmana 4
5
a Laboratory of Parasitic Diseases, NIAID, NIH, Bethesda, MD, USA 6
b Laboratory of Proteomics and Analytical Technologies, Advanced Technology 7
Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD, USA 8
c Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, 9
San Diego, CA, USA. 10
d Molecular Parasitology, Lindsley F. Kimball Research Institute, New York Blood 11
Center, New York, NY, USA 12
13
*Running title: Brugia malayi L3 to L4 development 14
15
To whom correspondence should be addressed: 16
Sasisekhar Bennuru, Laboratory of Parasitic Diseases, National Institute of Allergy and 17
Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 18
email: [email protected] 19
Keywords: Brugia malayi; cathepsin; cysteine protease; development; molting; 20
proteomics 21
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Brugia malayi L3 to L4 development
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Abstract 22
The establishment of infection with the lymphatic dwelling filarial parasites is 23
dependent on the infectivity and subsequent development of the infective larvae (L3) 24
within the human host to later stages (L4, adults) that require several developmental 25
molts. The molecular mechanisms underlying the developmental processes in parasitic 26
nematodes are not clearly defined. We report the proteomic profiles throughout the 27
entire L3 to L4 molt using an established in vitro molting process for the human 28
pathogen B. malayi. A total of 3466 proteins of B. malayi and 54 from Wolbachia were 29
detected at one or more time points. Based on the proteomic profiling, the L3 to L4 30
molting proteome can be broadly divided into an early, middle and late phase. 31
Enrichment of proteins, protein families and functional categories between each time 32
point or between phases primarily relate to energy metabolism, immune evasion 33
through secreted proteins, protein modification, and extracellular matrix-related 34
processes involved in the development of new cuticle. Comparative analyses with 35
somatic proteomes and transcriptomes highlighted the differential usage of cysteine 36
proteinases (CPLs), BmCPL-1, -4 and -5 in the L3-L4 molt compared to the adults and 37
microfilariae. Inhibition of the CPLs effectively blocked the in-vitro L3 to L4 molt. Overall, 38
only 4 Wolbachia proteins (Wbm0495, Wbm0793, Wbm0635, and Wbm0786) were 39
detected across all time points and suggest that they play an inconsequential role in the 40
early developmental process. 41
Importance 42
The neglected tropical diseases of lymphatic filariasis, onchocerciasis (or river 43
blindness), and loiasis are the three major filarial infections of humans that cause long-44
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Brugia malayi L3 to L4 development
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term disability, impaired childhood growth, reduced reproductive capacity. Global efforts 45
to control and/or eliminate these infections as a public health concern are based on 46
strategies and tools to strengthen the diagnostics, therapeutic and prophylactic 47
measures. A deeper understanding of the genes, proteins and pathways critical for the 48
development of the parasite is needed to help further investigate the mechanisms of 49
parasite establishment and disease progression, because not all the transmitted 50
infective larvae get to develop successfully and establish infections. The significance of 51
this study is in identifying the proteins and the pathways that are needed by the parasite 52
for successful developmental molts, that in turn will allow for investigating targets of 53
therapeutic and prophylactic potential. 54
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Brugia malayi L3 to L4 development
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Introduction 55
Lymphatic filariasis is caused primarily by the parasitic nematodes Wuchereria 56
bancrofti, Brugia malayi and Brugia timori. Infection is initiated when infective L3 larvae 57
enter the human host through the skin and subsequently develop into L4 after a 58
developmental molt. Given that the L3 to L4 molting process occurs across all 59
nematodes and that it is the essential first step towards establishing infection, the 60
molting process has been postulated to be a target for intervention strategies (1). 61
Despite the notion that these early (L3 and L4) developmental stages would be an 62
important target for prophylactic vaccines, the biology of these early mammalian stages 63
of the lymph-dwelling filarial parasites have been not well-studied. 64
There is evidence that molting and ecdysis in most nematodes are under the 65
control of neurosecretory and endocrine processes (2). While functional nuclear 66
hormone receptors (3, 4), have been identified in filarial nematodes and shown to 67
influence embryogenesis (5), it is not clear if they play any role during the molting 68
process. A number of enzymes including cathepsins, collagenases, lipases, Zn-69
metalloproteases and aminopeptidases have been implicated in the ecdysis of L3 larvae 70
(6-8). Transcriptional data from microarrays indicated that the transition of the L3 from 71
the vector (at ambient temperature) to the mammalian host (37°C) involves the 72
induction of expression of a wide variety of genes termed adaptation- and infectivity-73
associated genes (9). While the depletion of the endosymbiotic bacterium Wolbachia 74
by antibiotics results in sterility of adult female parasites and disrupts larval molting (10), 75
the significance of this symbiosis, however, in the molting process is not clear as filarial 76
(and other) nematodes that are Wolbachia-free also molt quite successfully. 77
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Because this developmental process can be recapitulated readily in vitro, in 78
serum-free conditions where the metabolic requirements and signals necessary for the 79
induction of the L3 to L4 molt have been defined (11, 12), we assessed the proteomic 80
profiles of the L3-L4 molt to understand molecularly the initiation of the molting process, 81
and subsequently identify and target specific crucial pathways that could prevent 82
parasite development. In the process, we also defined the protein expression profiles of 83
the endosymbiont, Wolbachia (wBm). 84
85
Results 86
B. malayi in vitro molting protein atlas. 87
The in vitro developmental molt of B. malayi from the infective L3 larvae to the L4 88
stage was partitioned into 9 segments (see Materials and Methods, Figure 1A) and 89
analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). At a false 90
discovery rate (FDR) of 0.01, a total of 3466 proteins of B. malayi (Table S1) and 57 of 91
Wolbachia (wBm) origin were identified (Table S2). Comparative expression profiles of 92
Brugia-derived proteins of the quadruplicates across all the time-points using Spearman 93
rank correlations revealed two distinct groups (Figure 1B), an early phase (L3, 3Hrs 94
and 24Hrs) and a mid-late phase, the latter being able to be further divided into an early 95
ascorbic or middle phase (5 days to 24HrAsc) and late phase (48HrAsc to L4). Principal 96
component analyses further highlight the changes in protein expression profiles 97
between the ascorbic phases (5 Days, 3HrAsc, 24HrAsc, 48HrAsc), Molting and L4 98
stages (Figure 1C). 99
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On average ~1500 proteins were identified from each stage by at least one 100
unique peptide (Table S1). The proteins were identified either exclusively in one stage, 101
commonly or randomly expressed across the stages, or at specific periods during the 102
developmental molt. The identified proteins were placed into defined clusters using t-103
SNE dimensionality reduction (13). This approach highlights additional sub-clusters of 104
proteins that in turn define transitions between early-mid, mid-late, common and random 105
expression (Figure S1-A). For example, the ‘early’ group comprised of three distinct 106
sub-groups that define the stages (L3, 3Hrs 24Hrs) during which sets of proteins were 107
expressed at high levels. Though the vast majority of proteins were detected in most 108
stages, certain clusters of these common proteins were observed to be present in 109
higher abundance across each of the stages (Figure S1-B). 110
Because the protein expression profile appeared to be broadly set into the early, 111
middle and late phases, the expression data were also visualized using supraHex (14) a 112
self-organizing and visualization tool (Figure 2). Protein clusters during early 113
development (3Hrs to 5Days) were shown by increased expression that included 114
cathepsin-L like cysteine proteases (Bm7675, Bm7676, Bm7677, Bm7679, Bm7681), 115
cystatin (Bm366), SCP-like extracellular protein (Bm4233) and conserved secreted 116
proteins (Bm16893, Bm16894, Bm16896) among others (15). The later phases of 117
molting were reflected by increased expression of enzymes and proteins involved in 118
cuticle synthesis. The comparisons of the enriched GO categories of the differentially 119
expressed proteins plotted by semantic similarity (Figure S2) between early, middle and 120
late phases highlight early cysteine-type peptidase catalytic activity (in Early <-> Middle; 121
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Early <-> Late), oxidoreductase activity, steroid dehydrogenase activity and peroxidase 122
and protein-disulfide oxidoreductase activities (in Middle <-> Late). 123
124
Functional enrichment 125
Because not all proteins can be classified through GO categories, B. malayi 126
proteins identified during the molting process were classified into functional groups (16, 127
17). The distribution of the functional groups (plotted as percentage of proteins identified 128
from each stage) again appears to be clustered into three broad groups (early, middle 129
and late) (Figure 3A, Supplemental Figure S3A, B). Along similar lines, gene set 130
enrichment analysis indicated enrichment for secreted and energy metabolism-related 131
proteins during the early phase (L3, 3Hrs and 24Hrs) compared to the later stages 132
(middle and late). While the energy metabolism-related proteins comprised of 133
dehydrogenases and oxidoreductases, the secreted class of proteins were primarily the 134
cysteine proteases, abundant larval transcripts, serpins, cystatins and several 135
conserved hypothetical proteins. 136
In contrast, enrichment (FDR < 0.01) of proteins involved in lipid metabolism, 137
protein export and protein modification were observed during the mid-to-late phase 138
(5days to L4), compared to the early phase (L3, 3Hrs and 24Hrs) (Supplemental 139
Figure S3B, C). The addition of ascorbic acid resulted in the enrichment of secreted 140
proteins (Supplemental Figure S3D) that were distinct from the secreted proteins 141
during the early phase (Figure 3) and annotated as conserved hypothetical proteins 142
and extracellular matrix-related proteins primarily composed of collagens and related 143
machinery. 144
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145
Stage-specific expression of cysteine proteinases 146
Phylogenetic analyses of all the B. malayi encoded cysteine proteases indicated 147
that the majority of cathepsin L-like cysteine proteases detected during the L3 to L4 148
developmental molt were similar to BmCPL-1, BmCPL-4 and BmCPL-5 (Figure 4A), 149
and very much similar to what has been observed in O. volvulus (18), B. pahangi (19) 150
and D. immitis (20). Cathepsin A (Bm3985, Bm6297), Cathepsin B (Bm2365), and 151
Cathepsin F (Bm1575, Bm3996) were increased during the ascorbic acid phase. 152
Interestingly, compared to the enrichment of CPL-1, -4 and -5 observed in the L3 to L4 153
stages, combined analysis with the protein profiles of the stage-specific somatic 154
proteomes (16) and transcriptomes (21) indicated the stage-specific expression and/or 155
utilization of Bm12799, Bm12798, Bm12797, Bm8207, Bm8172, Bm748 and Bm99 156
(CPL-2, -3, -6 and -7 like) by the immature and mature stages of microfilariae (Figure 157
4B). 158
Since the cathepsin’s have been hypothesized to be stored in the granules of the 159
glandular esophagus and transported through pseudocoelomic fluid networks and 160
secretory vessels to the hypodermis during molting(18), the cathepsin activity was 161
localized using ProSense 680, a fluorescent catabolic substrate of cathepsin at 24hrs 162
and 5days. As shown in Figure 5A cathepsin activity was more pronounced near the 163
hypodermal and sub-cuticular areas at day 5 compared to that seen in the area around 164
the gut/pharynx at 24hrs. As observed previously with other filarial parasites, inhibition 165
studies of cathepsins with chemical inhibitors were carried out to assess the impact of 166
cathepsins in this in-vitro molting model (19, 22). Similar to the studies with other filarial 167
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parasites, a dose-dependent inhibition of molting with Z-Phe-Ala-FMK was observed 168
(Figure 5B). In addition, a novel cysteine protease inhibitor - K11777 was much more 169
effective than Z-Phe-Ala-FMK, and at concentrations above 2 µM, blocked the 170
development of L3 larvae in vitro and completely killed all the larvae within 24hrs 171
(Figure 5C). At 1 µM K11777 larvae were still viable at 4 days but failed to molt. 172
173
Kinases 174
Ascorbic acid is known to regulate a wide variety of biochemical processes, of 175
which the activation/inhibition of kinases and collagens are most notable (23-26). To 176
understand the effect of ascorbic acid in inducing the crucial developmental molt, the 177
expression of the B. malayi kinome across the L3-L4 development was analyzed. A total 178
of 426 eukaryotic protein kinases (ePK) were identified, comprising 2.5% of the 179
predicted proteome of B. malayi. It appears that B. malayi may be missing the core 180
kinases CAMK/RAD53 and CMGC/RCK/MAK. Interestingly, the predominant clustering 181
of the kinases and their relative abundance upon stimulation with ascorbic acid appears 182
to activate and induce the expression of CAMK and AGC family of kinases (Figure 6), 183
that are known to regulate cytoskeletal reorganization and extracellular matrix 184
remodeling, and are potential therapeutic targets (27, 28). 185
186
Cuticle, Collagens and Collagen-machinery 187
Clustering of representative members of C. elegans collagens (29) with B. malayi 188
collagens detected during the molting process highlighted the specific expression of the 189
dpy-2, dpy-10, dpy-7 and dpy-8 and select Group-2 collagens during the middle phase 190
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(Figure 7A, B). The clustering data also indicate that constitutively expressed collagens 191
primarily belong to Group-1 collagens (Figure 7B). 192
The synthesis and trimerization of collagen fibers is a series of complex 193
enzymatic processes involving prolyl-4 hydroxylases, disulfide isomerases, peptidyl-194
prolyl cis-trans isomerase, blisterases and aminopeptidases (30). The clustering of the 195
collagen machinery proteins identified three well-defined clusters (Figure S4). Though 196
the exact combination of molecules involved in this process is unknown, it is likely that 197
collagen machinery components upregulated at 5-days and beyond are responsible for 198
the generation of the new cuticle. Surprisingly, even though nuclear hormone receptors 199
are known to play a role in molting, there was barely any expression of the nuclear 200
hormone receptors of B. malayi identified at the protein level. 201
202
Discussion 203
To establish a filarial infection in a vertebrate host, the infective L3 larvae must 204
undergo a series of successful developmental molts in the host to become adult 205
parasites. The B. malayi L3 to L4 molting in a controlled in vitro environment devoid of 206
serum or growth factors appears to be the best way to investigate the essential pathway 207
for the early development of the mammalian stage parasites. It should be noted that the 208
L3 larvae had to be shipped post harvesting and hence the proteomic profile might be 209
slightly different if they were to be cultured immediately upon harvest. Although gene 210
expression data of developmental stages in C. elegans (31, 32) and microarray 211
analyses of B. malayi L3 larvae (9) are available, direct comparative analyses of the in 212
vitro molting model data were limited, due to the variable durations of molting processes 213
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(relatively short developmental time in C. elegans (~9 hours) compared to B. malayi (~9 214
days) and the fact that unlike C. elegans, the L3’s of B. malayi cannot be synchronized. 215
Further, microarray analyses of B. malayi L3 larvae (9), were also not directly 216
comparable because of experimental design constraints. It is also to be noted that all 217
the transcriptomic and proteomics analyses are based on the quality of the annotated 218
genomes available. 219
The early phase of the L3 developmental process showed that cysteine 220
proteases play an important role during molting, a finding with parallels to studies in O. 221
volvulus (33). While the proteomic findings corroborate previous RNA expression data 222
of CPL-1, -4 and -5 (Group Ia) in the L3 stages, it was interesting to note that the 223
cysteine proteases (CPL-2, -3, -6, -7 and -8) that belong to group Ic (19) were most 224
abundant in the microfilarial and intra-uterine microfilarial stages. It has been 225
hypothesized that the CPLs in the L3s are stored in the granules of the glandular 226
esophagus that are transported during molting through pseudocoelomic fluid networks 227
and secretory vessels to the hypodermis and the cuticle (18). Fluorescent substrate 228
catalysis imaging demonstrated cathepsin-like activity in the granules of the esophageal 229
tube as early as 24 hours at 37°C and that remains visible in the hypodermal and sub-230
cuticular regions of the worm following ascorbic acid induction of molting in vitro. 231
Cystatins, are endogenous cysteine protease inhibitors and when transported to 232
the cuticles of the filarial L3 and L4 larvae during molting (18, 34), they possibly regulate 233
the cysteine protease activity that does not inhibit the formation of the new cuticle, but 234
rather support the separation of the L3 and L4 cuticles in vitro (18). The ability to inhibit 235
molting and/or development of B. malayi L3 larvae by chemical inhibitors of cathepsins 236
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(Z-Phe-Ala-FMK & K11777) demonstrate the critical role these molecules play in the 237
development of the nematode parasite. K11777, a novel cysteine protease inhibitor has 238
been shown to be effective against Trypanosoma cruzi, Leishmania tropica and 239
Schistosoma mansoni (35). However, although inhibition studies by RNAi have 240
previously been used with B. malayi (22, 36-38) and O.volvulus (33), B. malayi L3 241
larvae were quite sensitive to dsRNA, siRNA, shRNA targeting the cysteine proteases, 242
as the induction of molting with ascorbic acid in the presence of even non-specific 243
silencing nucleic acids resulted in death of all larvae. Likewise, although electroporation 244
has been used successfully in adult worms (37), it was lethal to B. malayi L3 larvae. 245
246
The middle and late phases of molting (Day 5 and beyond) were primarily 247
comprised of enzymes and proteins related to cuticle formation. Among the enzymes 248
were protein kinases that are known to be regulated by the diverse biochemical 249
functions of ascorbate and dehydroascorbate and influence the cytoskeletal 250
reorganization. For example, ROK1, MRCKa, AKT3/PKBa facilitate extracellular matrix 251
(ECM) remodeling, while the NIMA-related kinases NEKL-2/NEK8/NEK9 and NEKL-252
3/NEK6/NEK7, together with their ankyrin repeat partners, MLT-2/ANKS6, MLT-253
3/ANKS3, and MLT-4/INVS, are essential for normal molting (25). The NIMA-related 254
kinase network functionally interacts with CDC-42 and SID-3/ACK1 to facilitate ECM 255
remodeling. It would be interesting to investigate further the role of CAML and AGC 256
family of kinases expressed post ascorbic acid in the molting process. Ascorbic acid 257
also promotes the expression of procollagen C-proteinase enhancer (PCPE-1) (39), 258
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necessary for collagen fibril assembly. Though the timing was not as expected, the B. 259
malayi orthologue of PCPE (Bm3917) was detected at 48hr post-ascorbic acid. 260
Nematodes do have an ascorbate biosynthetic pathway that is significantly 261
different from that found in animals, plants and fungi (40). Although, the homologues of 262
the mammalian nucleobase ascorbate transporter or nucleobase cation symport 2 263
(NAT/NCS2; SCVT1 and SCVT2) (41) that support the active transport are not present 264
in B. malayi, the conserved NAT motif [QEP]NXGXXXXT[RKG] could be mapped to 265
Bm2885. However, Bm2885 protein was not detected in any of the stages. 266
Temporal regulation of the collagen gene expression in C. elegans has been 267
shown to occur during the molting period (42, 43). The expression patterns of various 268
classes of collagens were expressed at varying levels across the various stages or 269
specifically enriched in stage-specific molts (44, 45). Though the cuticles of larval and 270
adult stages appear to be similar both structurally and biochemically (46-48), the 271
preferential utilization of collagens by L3 larvae that cluster with Group 2 collagens (29) 272
corroborates data previously described (49, 50). The production of collagen influenced 273
by ascorbate in humans has been largely attributed (primarily as a co-factor) to prolyl 274
hydroxylation (51) and/or increases in steady-state levels of procollagen mRNA (52). 275
However, other studies suggested that the role of ascorbate in collagen synthesis may 276
be unrelated to hydroxylation (53). In addition, there have been other studies that have 277
suggested that this process may reflect a lack of transcriptional regulation (54) or may 278
involve the protein synthesis machinery or other mechanisms (38, 55). 279
Complementation of B. malayi encoded collagen enzymatic machinery genes in C. 280
elegans suggested both conserved and divergent functional activities (reviewed in (30)). 281
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The association between the filarial parasites and their endosymbiont Wolbachia 282
(wBm) is ancient (in evolutionary terms) with mutual, symbiotic interrelationships (56). 283
Data from studies targeting Wolbachia with antibiotics not only supports this symbiotic 284
relationship, but they also highlight the dependence of the filariae on the bacteria for a 285
diverse range of biological and stage-specific processes (57). Although B. malayi does 286
not have a complete gene set required for the de novo purine synthesis and may be 287
dependent on wBm, we were unable to observe this directly for the reason that the 288
culture medium was supplemented with ribonucleosides and deoxyribonucleosides. 289
Likewise, the influence of Wolbachia in the molting process was not clear in the molting 290
process. Although a total of 57 Wolbachial proteins were detectable, there was no 291
obvious phase-specific discernible patterns (Supplementary Figure S5), except for 292
Chaperonin GroEL (HSP60; Wbm0350) that was detected during the molting and L4 293
stages only. Only 4 Wolbachia proteins (Wbm0495-molecular chaperone DnaK, 294
Wbm0793 – Type IV secretory pathway VirB6 components, Wbm0635 – RecG-like 295
helicase, Wbm0786 – valyl-tRNA synthetase) were detected across each of the time 296
points. Though non-Wolbachia containing filarial parasites (e.g. Loa loa), having similar 297
genomic structures to those of B. malayi and W. bancrofti (58) and have normal L3-L4 298
molts, indicates no active role for Wolbachia in the molting process. It is likely, however, 299
that the low coverage of Wolbachia-derived peptides was influenced by the limitations 300
associated with mass spectrometry and highly limited wBm numbers and protein 301
content during the L3-L4 transition. Wolbachia can be depleted by antibiotics belonging 302
to the tetracycline class of antibiotics and causes sterilization of the adult females and 303
blocks development of the parasites. Because the in vitro molting efficacy was 304
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significantly inhibited by tetracycline, and by a chemically modified tetracycline (that 305
lacks anti-microbial activity) in the absence of Wolbachia clearance suggested 306
disruption of filarial physiology as a possible mechanism (59, 60). 307
In conclusion, using a defined in-vitro model of filarial molting that limits the 308
possibility of any extraneous or unknown mediators influencing the molting process our 309
data highlights the minimal set of proteins and processes needed for the B. malayi L3 310
larvae to molt to L4. Moreover, a number of these are very good targets for prophylactic 311
and/or therapeutic intervention, in other non-human filarial parasites. 312
313
MATERIALS AND METHODS 314
Parasites and molting model 315
B. malayi L3 larvae were obtained under contract from the FR3 facility at University of 316
Georgia to the NIAID. The L3 larvae were cultured (Figure 1A) as described previously 317
(61). The larvae were snap frozen immediately upon processing (‘BmL3’; D0), post-318
incubation at 37°C for 3 hrs (‘3Hrs’), 24 hrs (‘24Hrs’; D1), 5 days (‘5Days’; D5). 319
Following the addition of ascorbic acid (75 µM) at day 5, parasites were collected 3 hrs 320
later (‘3HrAsc’), 24 hrs later (‘24HrAsc’), and 48 hrs later (‘48HrAsc’) and then again 321
during molting (‘Molting’; Days 8/9) and finally as viable L4 larvae (‘BmL4’). The animal 322
procedures used for the life-cycle of B. malayi were conducted in accordance with the 323
animal care and use committee guidelines at the National Institutes of Health and at the 324
University of Georgia. 325
326
Protein extraction and Mass Spectrometry 327
and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105
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Total soluble proteins were extracted from ~500 larvae from each time point using the 328
UPX universal protein extraction kit (Protein Discovery, San Diego, CA) as per the 329
manufacturer’s instructions. The protein concentrations were estimated using Pierce 330
BCA protein assay kits. The protein samples from L3 to L4 larvae stage were reduced, 331
alkylated and trypsin digested overnight following filter-aided digestion procedure using 332
a FASP digestion kit (Protein Discovery, San Diego, CA). Tryptic peptides were further 333
desalted using C18 spin columns (Thermo Fisher Scientific, IL). Samples were then 334
lyophilized and reconstituted in 0.1% trifluoroacetic acid to be analyzed in 335
quadruplicates without fractionation for quantitation purposes. 336
Nanobore RPLC-MSMS was performed using an Agilent 1200 nanoflow LC 337
system coupled online with LTQ Orbitrap Velos mass spectrometer. The RPLC column 338
(75 µm i.d. x 10cm) were slurry-packed in-house with 5 µm, 300Å pore size C-18 339
stationary phase into fused silica capillaries with a flame pulled tip. After sample 340
injection, the column was washed for 20 min with 98% mobile phase A (0.1% formic 341
acid in water) at 0.5 µl/min. Peptides were eluted using a linear gradient of 2% mobile 342
phase B (0.1% formic acid in ACN) to 35% B in 100 minutes, then to 80% B over an 343
additional 20 minutes. The column flow-rate was maintained at 0.25 µl/min throughout 344
the separation gradient. The mass spectrometer was operated in a data-dependent 345
mode in which each full MS scan was followed by sixteen MS/MS scans wherein the 346
sixteen most abundant molecular ions were dynamically selected for collision-induced 347
dissociation (CID) using a normalized collision energy of 35%. 348
The LC-MS/MS data were processed using PEAKS 7 Studio (ver 7, 349
Bioinformatics Solutions Inc.). MS/MS data were searched against a combined decoy 350
and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105
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Brugia malayi L3 to L4 development
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database of B. malayi (http://parasite.wormbase.org; WBPS10) and Wolbachia (wBm) 351
containing both forward and reverse sequences as well as a common contaminant 352
database using default parameters. Dynamic modifications of methionine oxidation and 353
N-terminal acetylation as well as fixed modification of carbamidomethyl cysteine were 354
included in the database search. Only tryptic peptides with up to two missed cleavage 355
sites with a minimum peptide length of six amino acids were allowed. The false 356
discovery rate (FDR) was set to 0.01 and threshold-based filtering of -10logP scores of 357
30 for both peptide and protein identifications. Statistical tests were performed on 358
normalized spectral abundance (NSAF, relative abundance) of the proteins to determine 359
significant protein changes between time points (Table S1). Rarefaction analyses were 360
carried out using the number of peptides identified in each sample (Supplemental 361
Figure S6). The data were analyzed using JMP Genomics 9.0 (SAS Institute Inc) and R 362
(3.6). 363
364
Differential Analysis: 365
Gene Set Enrichment Analysis (GSEA, Broad Institute), a method for analyzing 366
molecular profiling data, examines the clustering of a pre-defined group of genes or 367
proteins (gene set) across the entire database in order to determine whether the gene 368
set has biased expression in one condition (or stage) versus another (62). For this 369
analysis, the entire list of B. malayi L3 larval molting-associated proteins were sorted on 370
their relative abundance. The distribution of proteins from an a priori defined set 371
throughout this ranked list was then determined using GSEA (as described previously 372
and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105
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Brugia malayi L3 to L4 development
18
(63)). Sets of genes encoding for proteins in each functional category were analyzed 373
using GSEA for specific enrichment of genes/proteins. 374
Differential expression of proteins between early, middle and late phases of the 375
molting process and also the early, middle or late phases with the other two phases 376
were analyzed by QPROT(64), an extension of QSPEC(65) suite for label free 377
proteomics. Gene Ontology enrichment for the differentially expressed proteins was 378
analyzed using R implementation of TopGO (66) and semantic similarity graphed using 379
REVIGO (67). 380
381
Cathepsin Activity and Chemical inhibition: 382
Prosense 680 Fluorescent Imaging Agent (Perkin Elmer #NEV10003, previously 383
VISEN) was used to visualize the activity of cathepsin’s in vivo after incubating the 384
larvae for various time-points at 37°C. Fluorescence images were collected on a Leica 385
SP5 X-WLL confocal microscope (Leica Microsystems, Exton, PA). Z-Phe-Ala-FMK 386
(Sigma # C1480) was prepared as 10 µM stocks in DMSO. L3 larvae were cultured in 387
the presence of 2 µM, 4 µM, 8 µM and 10 µM of Z-Phe-Ala-FMK in L3 media for 5 days. 388
The numbers of shed cuticles (as indicator of successful L3 larval molt) were 389
enumerated on Day 10. K11777, a novel drug that targets cysteine proteases (35) was 390
also tested at concentrations of 2 µM – 10 µM. 391
392
Kinome analyses 393
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Brugia malayi L3 to L4 development
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The kinome of B. malayi was analyzed using Kinannote (68) and the intensities 394
(expression abundance) of the kinases was plotted using a slightly modified version of 395
Kinomerender (69). 396
397
Sequence analysis 398
Sequences of the cysteine proteases, collagens (B. malayi and C. elegans) were 399
aligned in MegAlign Pro (DNASTAR, Lasergene 17) using MUSCLE algorithm. Next, 400
maximum likelihood (ML) trees and bootstrap (BS) trees were generated with a final 401
“best” tree generated from the best scoring ML and BS trees using RAxML v8.2.10. The 402
resulting phylogenetic trees were visualized in FigTree 1.4 (http://tree.bio.ed.ac.uk). 403
404
Acknowledgements 405
This work was funded in part by the Division of Intramural Research, National Institute 406
of Allergy and Infectious Diseases, National Institutes of Health. 407
and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105
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Brugia malayi L3 to L4 development
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References 408
1. Lustigman S. 1993. Molting, enzymes and new targets for chemotherapy of Onchocerca 409
volvulus. Parasitol Today 9:294-7. 410
2. Lee DL. 2002. The Biology of Nematodes. CRC Press, New York. 411
3. Tzertzinis G, Egana AL, Palli SR, Robinson-Rechavi M, Gissendanner CR, Liu C, Unnasch 412
TR, Maina CV. 2010. Molecular evidence for a functional ecdysone signaling system in 413
Brugia malayi. PLoS neglected tropical diseases 4:e625. 414
4. Shea C, Richer J, Tzertzinis G, Maina CV. 2010. An EcR homolog from the filarial parasite, 415
Dirofilaria immitis requires a ligand-activated partner for transactivation. Molecular and 416
biochemical parasitology 171:55-63. 417
5. Mhashilkar AS, Adapa SR, Jiang RH, Williams SA, Zaky W, Slatko BE, Luck AN, Moorhead 418
AR, Unnasch TR. 2016. Phenotypic and molecular analysis of the effect of 20-419
hydroxyecdysone on the human filarial parasite Brugia malayi. Int J Parasitol 420
doi:10.1016/j.ijpara.2016.01.005. 421
6. Gamble HR, Purcell JP, Fetterer RH. 1989. Purification of a 44 kilodalton protease which 422
mediates the ecdysis of infective Haemonchus contortus larvae. Molecular and 423
biochemical parasitology 33:49-58. 424
7. Rogers WP. 1982. Enzymes in the exsheathing fluid of nematodes and their biological 425
significance. Int J Parasitol 12:495-502. 426
8. Rogers WP, Brooks F. 1976. Zinc as a co-factor for an enzyme involved in exsheathment 427
of Haemonchus contortus. International journal for parasitology 6:315-9. 428
and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105
The copyright holder for this preprint (whichthis version posted May 6, 2021. ; https://doi.org/10.1101/2021.05.06.439182doi: bioRxiv preprint
Brugia malayi L3 to L4 development
21
9. Li BW, Rush AC, Mitreva M, Yin Y, Spiro D, Ghedin E, Weil GJ. 2009. Transcriptomes and 429
pathways associated with infectivity, survival and immunogenicity in Brugia malayi L3. 430
BMC Genomics 10:267. 431
10. Hoerauf A, Nissen-Pahle K, Schmetz C, Henkle-Duhrsen K, Blaxter ML, Buttner DW, Gallin 432
MY, Al-Qaoud KM, Lucius R, Fleischer B. 1999. Tetracycline therapy targets intracellular 433
bacteria in the filarial nematode Litomosoides sigmodontis and results in filarial 434
infertility. The Journal of Clinical Investigation 103:11-8. 435
11. Rajan TV, Paciorkowski N, Kalajzic I, McGuiness C. 2003. Ascorbic acid is a requirement 436
for the morphogenesis of the human filarial parasite Brugia malayi. The Journal of 437
parasitology 89:868-70. 438
12. Ramesh M, McGuiness C, Rajan TV. 2005. The L3 to L4 molt of Brugia malayi: real time 439
visualization by video microscopy. The Journal of parasitology 91:1028-33. 440
13. van der Maaten L. 2013. Barnes-Hut-SNE. arXiv:13013342v1 [csLG] 441
14. Fang H, Gough J. 2014. supraHex: an R/Bioconductor package for tabular omics data 442
analysis using a supra-hexagonal map. Biochem Biophys Res Commun 443:285-9. 443
15. Maizels RM, Blaxter ML, Scott AL. 2001. Immunological genomics of Brugia malayi: 444
filarial genes implicated in immune evasion and protective immunity. Parasite Immunol 445
23:327-44. 446
16. Bennuru S, Meng Z, Ribeiro JM, Semnani RT, Ghedin E, Chan K, Lucas DA, Veenstra TD, 447
Nutman TB. 2011. Stage-specific proteomic expression patterns of the human filarial 448
parasite Brugia malayi and its endosymbiont Wolbachia. Proc Natl Acad Sci U S A 449
108:9649-54. 450
and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105
The copyright holder for this preprint (whichthis version posted May 6, 2021. ; https://doi.org/10.1101/2021.05.06.439182doi: bioRxiv preprint
Brugia malayi L3 to L4 development
22
17. Bennuru S, Semnani R, Meng Z, Ribeiro JM, Veenstra TD, Nutman TB. 2009. Brugia 451
malayi excreted/secreted proteins at the host/parasite interface: stage- and gender-452
specific proteomic profiling. PLoS Negl Trop Dis 3:e410. 453
18. Lustigman S, McKerrow JH, Shah K, Lui J, Huima T, Hough M, Brotman B. 1996. Cloning 454
of a cysteine protease required for the molting of Onchocerca volvulus third stage 455
larvae. J Biol Chem 271:30181-9. 456
19. Guiliano DB, Hong X, McKerrow JH, Blaxter ML, Oksov Y, Liu J, Ghedin E, Lustigman S. 457
2004. A gene family of cathepsin L-like proteases of filarial nematodes are associated 458
with larval molting and cuticle and eggshell remodeling. Molecular and biochemical 459
parasitology 136:227-42. 460
20. Richer JK, Hunt WG, Sakanari JA, Grieve RB. 1993. Dirofilaria immitis: effect of 461
fluoromethyl ketone cysteine protease inhibitors on the third- to fourth-stage molt. Exp 462
Parasitol 76:221-31. 463
21. Choi YJ, Ghedin E, Berriman M, McQuillan J, Holroyd N, Mayhew GF, Christensen BM, 464
Michalski ML. 2011. A deep sequencing approach to comparatively analyze the 465
transcriptome of lifecycle stages of the filarial worm, Brugia malayi. PLoS neglected 466
tropical diseases 5:e1409. 467
22. Ford L, Zhang J, Liu J, Hashmi S, Fuhrman JA, Oksov Y, Lustigman S. 2009. Functional 468
Analysis of the Cathepsin-Like Cysteine Protease Genes in Adult Brugia malayi Using RNA 469
Interference. PLoS Negl Trop Dis 3:e377. 470
and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105
The copyright holder for this preprint (whichthis version posted May 6, 2021. ; https://doi.org/10.1101/2021.05.06.439182doi: bioRxiv preprint
Brugia malayi L3 to L4 development
23
23. Carcamo JM, Pedraza A, Borquez-Ojeda O, Zhang B, Sanchez R, Golde DW. 2004. Vitamin 471
C is a kinase inhibitor: dehydroascorbic acid inhibits IkappaBalpha kinase beta. Mol Cell 472
Biol 24:6645-52. 473
24. Bowie AG, O'Neill LA. 2000. Vitamin C inhibits NF-kappa B activation by TNF via the 474
activation of p38 mitogen-activated protein kinase. J Immunol 165:7180-8. 475
25. Lazetic V, Joseph BB, Bernazzani SM, Fay DS. 2018. Actin organization and endocytic 476
trafficking are controlled by a network linking NIMA-related kinases to the CDC-42-SID-477
3/ACK1 pathway. PLoS Genet 14:e1007313. 478
26. Panowski SH, Dillin A. 2009. Signals of youth: endocrine regulation of aging in 479
Caenorhabditis elegans. Trends Endocrinol Metab 20:259-64. 480
27. Nawaratna SSK, You H, Jones MK, McManus DP, Gobert GN. 2018. Calcium and 481
Ca(2+)/Calmodulin-dependent kinase II as targets for helminth parasite control. 482
Biochem Soc Trans 46:1743-1751. 483
28. Leroux AE, Schulze JO, Biondi RM. 2018. AGC kinases, mechanisms of regulation and 484
innovative drug development. Semin Cancer Biol 48:1-17. 485
29. Johnstone IL. 2000. Cuticle collagen genes. Expression in Caenorhabditis elegans. 486
Trends in genetics : TIG 16:21-7. 487
30. Page AP, Stepek G, Winter AD, Pertab D. 2014. Enzymology of the nematode cuticle: A 488
potential drug target? Int J Parasitol Drugs Drug Resist 4:133-41. 489
31. Gerstein MB, Lu ZJ, Van Nostrand EL, Cheng C, Arshinoff BI, Liu T, Yip KY, Robilotto R, 490
Rechtsteiner A, Ikegami K, Alves P, Chateigner A, Perry M, Morris M, Auerbach RK, Feng 491
X, Leng J, Vielle A, Niu W, Rhrissorrakrai K, Agarwal A, Alexander RP, Barber G, Brdlik 492
and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105
The copyright holder for this preprint (whichthis version posted May 6, 2021. ; https://doi.org/10.1101/2021.05.06.439182doi: bioRxiv preprint
Brugia malayi L3 to L4 development
24
CM, Brennan J, Brouillet JJ, Carr A, Cheung MS, Clawson H, Contrino S, Dannenberg LO, 493
Dernburg AF, Desai A, Dick L, Dose AC, Du J, Egelhofer T, Ercan S, Euskirchen G, Ewing B, 494
Feingold EA, Gassmann R, Good PJ, Green P, Gullier F, Gutwein M, Guyer MS, Habegger 495
L, Han T, Henikoff JG, et al. 2010. Integrative analysis of the Caenorhabditis elegans 496
genome by the modENCODE project. Science 330:1775-87. 497
32. Wang J, Kim SK. 2003. Global analysis of dauer gene expression in Caenorhabditis 498
elegans. Development 130:1621-34. 499
33. Lustigman S, Zhang J, Liu J, Oksov Y, Hashmi S. 2004. RNA interference targeting 500
cathepsin L and Z-like cysteine proteases of Onchocerca volvulus confirmed their 501
essential function during L3 molting. Mol Biochem Parasitol 138:165-70. 502
34. Aboobaker AA, Blaxter ML. 2003. Use of RNA interference to investigate gene function 503
in the human filarial nematode parasite Brugia malayi. Molecular and biochemical 504
parasitology 129:41-51. 505
35. Singh M, Singh PK, Misra-Bhattacharya S. 2012. RNAi mediated silencing of ATPase RNA 506
helicase gene in adult filarial parasite Brugia malayi impairs in vitro microfilaria release 507
and adult parasite viability. Journal of biotechnology 157:351-8. 508
36. Landmann F, Foster JM, Slatko BE, Sullivan W. 2012. Efficient in vitro RNA interference 509
and immunofluorescence-based phenotype analysis in a human parasitic nematode, 510
Brugia malayi. Parasites & vectors 5:16. 511
37. Lustigman S, Brotman B, Huima T, Prince AM, McKerrow JH. 1992. Molecular cloning 512
and characterization of onchocystatin, a cysteine proteinase inhibitor of Onchocerca 513
volvulus. The Journal of biological chemistry 267:17339-46. 514
and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105
The copyright holder for this preprint (whichthis version posted May 6, 2021. ; https://doi.org/10.1101/2021.05.06.439182doi: bioRxiv preprint
Brugia malayi L3 to L4 development
25
38. Anonymous. Cysteine Proteases of Pathogenic Organisms, vol 712. Landes Bioscience 515
AND Springer Science. 516
39. Gohar O, Weiss T, Wineman E, Kessler E. 2019. Ascorbic Acid Promotes Procollagen C-517
Proteinase Enhancer 1 Expression, Secretion, and Cell Membrane Localization. Anat Rec 518
(Hoboken) doi:10.1002/ar.24182. 519
40. Patananan AN, Budenholzer LM, Pedraza ME, Torres ER, Adler LN, Clarke SG. 2015. The 520
invertebrate Caenorhabditis elegans biosynthesizes ascorbate. Arch Biochem Biophys 521
569:32-44. 522
41. Gournas C, Papageorgiou I, Diallinas G. 2008. The nucleobase-ascorbate transporter 523
(NAT) family: genomics, evolution, structure-function relationships and physiological 524
role. Mol Biosyst 4:404-16. 525
42. Cox GN, Kusch M, DeNevi K, Edgar RS. 1981. Temporal regulation of cuticle synthesis 526
during development of Caenorhabditis elegans. Developmental biology 84:277-85. 527
43. Politz JC, Edgar RS. 1984. Overlapping stage-specific sets of numerous small collagenous 528
polypeptides are translated in vitro from Caenorhabditis elegans RNA. Cell 37:853-60. 529
44. Cox GN, Hirsh D. 1985. Stage-specific patterns of collagen gene expression during 530
development of Caenorhabditis elegans. Molecular and cellular biology 5:363-72. 531
45. Kramer JM, Cox GN, Hirsh D. 1985. Expression of the Caenorhabditis elegans collagen 532
genes col-1 and col-2 is developmentally regulated. The Journal of biological chemistry 533
260:1945-51. 534
46. Cox GN, Kusch M, Edgar RS. 1981. Cuticle of Caenorhabditis elegans: its isolation and 535
partial characterization. The Journal of cell biology 90:7-17. 536
and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105
The copyright holder for this preprint (whichthis version posted May 6, 2021. ; https://doi.org/10.1101/2021.05.06.439182doi: bioRxiv preprint
Brugia malayi L3 to L4 development
26
47. Cox GN, Staprans S, Edgar RS. 1981. The cuticle of Caenorhabditis elegans. II. Stage-537
specific changes in ultrastructure and protein composition during postembryonic 538
development. Developmental biology 86:456-70. 539
48. Edgar RS, Cox GN, Kusch M, Politz JC. 1982. The Cuticle of Caenorhabditis elegans. 540
Journal of nematology 14:248-58. 541
49. Kingston IB, Pettitt J. 1990. Structure and expression of Ascaris suum collagen genes: a 542
comparison with Caenorhabditis elegans. Acta tropica 47:283-7. 543
50. Kingston IB, Wainwright SM, Cooper D. 1989. Comparison of collagen gene sequences in 544
Ascaris suum and Caenorhabditis elegans. Molecular and biochemical parasitology 545
37:137-46. 546
51. Anonymous. 1978. The function of ascorbic acid in collagen formation. Nutrition reviews 547
36:118-21. 548
52. Schwarz RI. 1985. Procollagen secretion meets the minimum requirements for the rate-549
controlling step in the ascorbate induction of procollagen synthesis. The Journal of 550
biological chemistry 260:3045-9. 551
53. Murad S, Grove D, Lindberg KA, Reynolds G, Sivarajah A, Pinnell SR. 1981. Regulation of 552
collagen synthesis by ascorbic acid. Proceedings of the National Academy of Sciences of 553
the United States of America 78:2879-82. 554
54. Jeffrey JJ, Martin GR. 1966. The role of ascorbic acid in the biosynthesis of collagen. II. 555
Site and nature of ascorbic acid participation. Biochimica et biophysica acta 121:281-91. 556
and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105
The copyright holder for this preprint (whichthis version posted May 6, 2021. ; https://doi.org/10.1101/2021.05.06.439182doi: bioRxiv preprint
Brugia malayi L3 to L4 development
27
55. Stanley P, Stein PE. 2003. BmSPN2, a serpin secreted by the filarial nematode Brugia 557
malayi, does not inhibit human neutrophil proteinases but plays a noninhibitory role. 558
Biochemistry 42:6241-8. 559
56. Fenn K, Blaxter M. 2004. Are filarial nematode Wolbachia obligate mutualist symbionts? 560
Trends Ecol Evol 19:163-6. 561
57. Newton ILG, Slatko BE. 2019. Symbiosis Comes of age at the 10(th) Biennial Meeting of 562
Wolbachia Researchers. Appl Environ Microbiol doi:10.1128/AEM.03071-18. 563
58. Desjardins CA, Cerqueira GC, Goldberg JM, Dunning Hotopp JC, Haas BJ, Zucker J, Ribeiro 564
JM, Saif S, Levin JZ, Fan L, Zeng Q, Russ C, Wortman JR, Fink DL, Birren BW, Nutman TB. 565
2013. Genomics of Loa loa, a Wolbachia-free filarial parasite of humans. Nat Genet 566
45:495-500. 567
59. Smith HL, Rajan TV. 2000. Tetracycline inhibits development of the infective-stage larvae 568
of filarial nematodes in vitro. Experimental parasitology 95:265-70. 569
60. Rajan TV. 2004. Relationship of anti-microbial activity of tetracyclines to their ability to 570
block the L3 to L4 molt of the human filarial parasite Brugia malayi. The American 571
journal of tropical medicine and hygiene 71:24-8. 572
61. Bennuru S, Semnani R, Meng Z, Ribeiro JM, Veenstra TD, Nutman TB. 2009. Brugia 573
malayi excreted/secreted proteins at the host/parasite interface: stage- and gender-574
specific proteomic profiling. PLoS neglected tropical diseases 3:e410. 575
62. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, 576
Pomeroy SL, Golub TR, Lander ES, Mesirov JP. 2005. Gene set enrichment analysis: a 577
and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105
The copyright holder for this preprint (whichthis version posted May 6, 2021. ; https://doi.org/10.1101/2021.05.06.439182doi: bioRxiv preprint
Brugia malayi L3 to L4 development
28
knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl 578
Acad Sci U S A 102:15545-50. 579
63. Bennuru S, Meng Z, Ribeiro JM, Semnani RT, Ghedin E, Chan K, Lucas DA, Veenstra TD, 580
Nutman TB. 2011. Stage-specific proteomic expression patterns of the human filarial 581
parasite Brugia malayi and its endosymbiont Wolbachia. Proceedings of the National 582
Academy of Sciences of the United States of America 108:9649-54. 583
64. Choi H, Kim S, Fermin D, Tsou CC, Nesvizhskii AI. 2015. QPROT: Statistical method for 584
testing differential expression using protein-level intensity data in label-free quantitative 585
proteomics. J Proteomics 129:121-126. 586
65. Choi H, Fermin D, Nesvizhskii AI. 2008. Significance analysis of spectral count data in 587
label-free shotgun proteomics. Mol Cell Proteomics 7:2373-85. 588
66. Alexa A, Rahnenfuhrer J. 2016. topGO: Enrichment Analysis for Gene Ontology. R 589
package version 2300. 590
67. Supek F, Bosnjak M, Skunca N, Smuc T. 2011. REVIGO summarizes and visualizes long 591
lists of gene ontology terms. PLoS One 6:e21800. 592
68. Goldberg JM, Griggs AD, Smith JL, Haas BJ, Wortman JR, Zeng Q. 2013. Kinannote, a 593
computer program to identify and classify members of the eukaryotic protein kinase 594
superfamily. Bioinformatics 29:2387-94. 595
69. Chartier M, Chenard T, Barker J, Najmanovich R. 2013. Kinome Render: a stand-alone 596
and web-accessible tool to annotate the human protein kinome tree. PeerJ 1:e126. 597
598
and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105
The copyright holder for this preprint (whichthis version posted May 6, 2021. ; https://doi.org/10.1101/2021.05.06.439182doi: bioRxiv preprint
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Figure Legends: 599
600
Figure 1. Overview of the B. malayi L3 molting proteome. A) The time line for the L3 601
to L4 molting, with days (inverted blue triangles) and the time-points profiled (red 602
triangles). B). Correlation heat map of quadruplicates from each time point. Red to blue 603
denotes higher to lower abundance of detected protein. The early and later (middle, 604
late) phases are highlighted with the black squares C). Three-dimensional principal 605
component analyses plot highlighting the proteomic signature profiles across the time 606
points of the molting process 607
608
Figure 2. Clusters of molting proteome. The supraHex maps illustrate sample-609
specific expression profile, where proteins with similar expression profile are mapped to 610
the same position or cell. The nine time-points are broadly placed into three phases 611
(early, middle and late) are shown. The proteins clustered in the highlighted cells are 612
shown to the right. The color bar codes for expression levels [log2(intensity)], from violet 613
to red denoting high to low expression. 614
615
Figure 3. Functional analysis of differentially expressed proteins. The line graphs 616
represent the significantly enriched proteins during the early, middle or late phases, 617
while the pie charts represent their functional classifications. The values on the x-axis 618
denotes the normalized expression levels 619
620
and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105
The copyright holder for this preprint (whichthis version posted May 6, 2021. ; https://doi.org/10.1101/2021.05.06.439182doi: bioRxiv preprint
Brugia malayi L3 to L4 development
30
Figure 4. Cysteine proteases of B. malayi. A). Maximum likelihood phylogenetic 621
analyses of the B. malayi cysteine proteases using RaxML under the conditions of the 622
GAMMA model with nodal support values generated through 1000 bootstrap replicates. 623
The tree depicts the proteases detected during the molting process (red), are primarily 624
BmCPL-1, -4 and -5 like cysteine proteases. The proteases with previous proteomic 625
and/or transcriptomic evidence in embryonic stages and microfilaria (blue) or 626
constitutively across all stages (violet), and known B. malayi cysteine proteases (black, 627
prefixed with BmCPL). B) Heatmap depicts the expression and clustering of the 628
cysteine proteases detected in the current study (green), with the stage-specific somatic 629
proteomes (blue) or stage-specific transcriptomes (Pink). The corresponding PubLocus 630
annotation is also provided as the somatic proteome and transcriptomes were based on 631
the previous annotation. 632
633
Figure 5. Cathepsin Activity. A) Confocal images showing the breakdown of 634
ProSense680 fluorescent substrate in the gut/pharyngeal tract at 24hrs and hypodermal 635
areas by day 5; B. Inhibition of cathepsin activity with z-Phe-Ala-FMK shows a dose 636
dependent (2 µM to 10 µM) inhibition of molting in L3 larvae at 10 days. C. Inhibition 637
and death of L3 larvae by the cathepsin inhibitor K11777, plotted as survival graph over 638
4 days. Each dot/time point represents the average of 10 wells with ~10 larvae in each 639
well. 640
641
Figure 6. The kinome of Brugia malayi during molting. The figure denotes the 642
kinases (annotated with B. malayi accession and kinase name) detected across the 643
and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105
The copyright holder for this preprint (whichthis version posted May 6, 2021. ; https://doi.org/10.1101/2021.05.06.439182doi: bioRxiv preprint
Brugia malayi L3 to L4 development
31
various time points during the molting process overlaid on the phylogenetic tree of 644
kinases with the major kinase family groups (CMGC, AGC, CAMK, STE, TKL, TK, 645
atypical protein kinases). The size of the circle denotes the intensity of expression. 646
Concentric rings denote expression in multiple stages at varying intensities. 647
648
Figure 7. Collagens and cuticle machinery. A) Parallel plots depicting clusters of 649
enhanced expression of collagen proteins upon induction with ascorbic acid during the 650
molting process. The vertical lines denote the quadruplicates at each time point B) 651
Phylogenetic tree based on C. elegans collagen groups (black), indicates preferential 652
utilization of group2 and ‘dpy’ like collagens during the ascorbic phase (violet), and a 653
more distributed constitutively expressed (orange) or during late phase (red). Collagen 654
proteins not detected are in grey. 655
656
Figure S1. Stage-specific proteomic expression. A) Two-dimensional t-SNE plot of 657
the protein abundance. Each point represents a protein colored by the cluster group B) 658
Circular polar histogram of protein abundance across the clusters defined by t-SNE. 659
The abbreviated pie names: EM – Early to Mid; L – Late; PM – Pre-Molting; Asc – 660
Ascorbic Phase; Asc1 – 3HrAsc; Asc2 – 24HrAsc; Asc3 – 48HrAsc; Molt – Molting. The 661
concentric circles represent the stage-specific proportion of the protein. C) Heatmap of 662
the proteins defined as ‘common’ by t-SNE, highlighting the increased abundance in 663
specific time-points. D) Heatmap of the proteins detected only during early, middle and 664
late phases. Red to blue denoted high to low abundance for both the heatmaps. 665
666
and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105
The copyright holder for this preprint (whichthis version posted May 6, 2021. ; https://doi.org/10.1101/2021.05.06.439182doi: bioRxiv preprint
Brugia malayi L3 to L4 development
32
Figure S2. GO enrichment. The scatterplot shows the cluster representatives (i.e. 667
terms remaining after the redundancy reduction) in a two-dimensional space derived by 668
applying multidimensional scaling to a matrix of the GO terms' semantic 669
similarities. Bubble color indicates the user-provided p-value (legend in upper right-hand 670
corner); size indicates the frequency of the GO term in the underlying GOA database 671
(bubbles of more general terms are larger). 672
673
Figure S3. Functional classification and enrichment. A) The heatmap depicts the 674
number of proteins classified into functional groups. Red to yellow denotes high to low. 675
B-D) The gene set enrichment graphs depicting the enriched state of secreted proteins 676
during early phase (B), lipid metabolism during the mid-late phase (C), and distinct 677
group of secreted proteins during the ascorbic acid phase (D). Left half of GSEA plots 678
shows the enrichment score with heat maps of the corresponding proteins from 679
quadruplicates from each set on the right. L3-24hrs set comprises of Fresh L3, 3Hr and 680
24Hr of culture at 37oC; 5Days-L4 set includes 5days, 3HrAsc, 24HrAsc and 48HrAsc, 681
molting and L4; Asc-Phase includes 3HrAsc, 24HrAsc and 48HrAsc. Red to blue 682
indicates higher to lower expression. 683
684
Figure S4. Collagens and cuticle machinery. Heatmap depicts the clustering of 685
proteins involved in the cuticle synthesis. Red to blue denotes high to low expression. 686
687
and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105
The copyright holder for this preprint (whichthis version posted May 6, 2021. ; https://doi.org/10.1101/2021.05.06.439182doi: bioRxiv preprint
Brugia malayi L3 to L4 development
33
Figure S5. Wolbachial (wBm) proteins. The heatmap shows the wolbachial proteins 688
identified during the L3-L4 developmental molt. Red to blue denotes high to low 689
expression. 690
691
Figure S6. Rarefaction Curves 692
Rarefaction curves as function of number of peptides identified across the time-points 693
(left panel), and the replicates within each time-point (right). 694
695
Table S1. Normalized spectral abundances of B. malayi proteins 696
The table lists the normalized spectral abundances of B. malayi proteins identified 697
during each of the time-points of the molting process. A, B, C and D represent 698
quadruplicates from each time-point. The significant changes observed between the 699
early, middle and late phases are represented by the log fold changes, false discovery 700
rate (FDR), signal to noise (STN) and the corresponding p-values. The functional 701
classification is also listed. 702
703
Table S2. Normalized spectral abundances of Wolbachia proteins 704
The table lists the normalized spectral abundances of Wolbachia proteins identified 705
during each of the time points of the molting process. A, B, C and D represent 706
quadruplicates from each time-point. The functional classification is also listed. 707
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The copyright holder for this preprint (whichthis version posted May 6, 2021. ; https://doi.org/10.1101/2021.05.06.439182doi: bioRxiv preprint
B C
Figure 1
A
BmL3
3Hr
24Hr
24HrAsc
3HrAsc
5Days
48HrAsc
Molting
BmL4
and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105
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BmL3 24Hrs
5Days 24HrAsc
48HrAsc BmL4
EARL
YM
IDDL
ELA
TE
Cathepsin L-like cysteine proteasesBm7675; Bm7676; Bm7677; Bm7679, Bm7681
Conserved secreted protein precursor (ALT proteins)Bm16893; Bm16894; Bm16896, Bm17586SCP-like extracellular protein - Bm4233bCystatin - Bm366Pyruvate dehydrogenase subunit beta - Bm7953Catalytically inactive chitinase-like lectin - Bm8301
Glutathione-S-transferase – Bm10857; Bm17385BMA-MLT-9 – Bm3215Vacuolar sorting protein VPS45/Stt10 (Sec1 family) – Bm13830Conserved secreted protein precursor – Bm17670; Bm3280Calmodulin – Bm17348
Excretory/secretory protein Juv-p120 precursor – Bm17718
Cystatin-type cysteine proteinase inhibitor CPI-2 – Bm10669
Collagen col-34 – Bm18057
Regulator of microtubule dynamics protein 1 – Bm2444
Cuticle collagen 13 precursor – Bm4507Collagenase NC10 and Endostatin family protein – Bm8092
Nematode cuticle collagen N-terminal domain containing protein – Bm9021
Prolyl oligopeptidase – Bm9037Bm-NOAH-2 – Bm4245
FKBP-type peptidyl-prolyl cis-trans isomerase – Bm3724
Leucyl aminopeptidase – Bm9816
Angiotensin I-converting enzyme – Bm2712
Figure 2
3Hrs
3HrAsc
Molting
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Figure 3
Early Middle Late
Early Middle Late
Early Middle Late
Early Middle Late
Early Middle Late
Early Middle Late
Nor
mal
ized
Expr
essio
n
Nor
mal
ized
Expr
essio
n
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The copyright holder for this preprint (whichthis version posted May 6, 2021. ; https://doi.org/10.1101/2021.05.06.439182doi: bioRxiv preprint
L3 3Hrs
24H
rs
5Day
3HrA
sc
24H
rAsc
48H
rAsc
Mol
ting
L4 AMSo
mAF
Som
MFS
om
L3So
m
UTM
F
EE_R
NAs
eq
IM_R
NAs
eqM
M_R
NAs
eq
L3_R
NAs
eq
L4_R
NAs
eqAM
_RN
Aseq
AF_R
NAs
eq
Bm1666a :: Bm1_02100 - cathepsin L-like cysteine proteinase
Bm7681 :: Bm1_20385 - cathepsin L-like cysteine proteinase
Bm7675 :: Bm1_53615 - cathepsin L-like cysteine proteinase
Bm7676 :: Bm1_20385 - cathepsin L-like cysteine proteinase
Bm7677 :: Bm1_20385 - cathepsin L-like cysteine proteinase
Bm7679 :: Bm1_53615 - cathepsin L-like cysteine proteinase
Bm2028a :: Bm1_21035 - Cytosolic Ca2+-dependent cysteine protease (calpain)
Bm2028b :: Bm1_21035 - Cytosolic Ca2+-dependent cysteine protease (calpain)Bm7154a :: Bm1_30345 - Cytosolic Ca2+-dependent cysteine protease (calpain)Bm7154b :: Bm1_30345 - Cytosolic Ca2+-dependent cysteine protease (calpain)
Bm3985a :: Bm1_43130 - Serine carboxypeptidases (lysosomal cathepsin A)Bm3985b :: Bm1_43130 - Serine carboxypeptidases (lysosomal cathepsin A)Bm6297 :: Bm1_43130 - Serine carboxypeptidases (lysosomal cathepsin A)
Bm2365 :: Bm1_33735 - cathepsin B-like cysteine proteinase
Bm1575 :: Bm1_17125 - cathepsin F-like cysteine proteinase partialBm3996 :: Bm1_17125 - cathepsin F-like cysteine proteinase partial
Bm12975 :: Bm1_36255 - Papain family cysteine protease
Bm12803 :: Bm1_18805 - cahepsin L-like cysteine protease
Bm12798a :: Bm1_06180 - cathepsin L-like cysteine proteinase
Bm12798b :: Bm1_06185 - cathepsin L-like cysteine proteinase
Bm9835 :: Bm1_36255 - Papain family cysteine protease
Bm12799 :: Bm1_06185 - Papain family cysteine protease containing proteinBm8172b :: Bm1_06185 - cathepsin L-like cysteine proteinaseBm99 :: Bm1_06185 - cathepsin L-like cysteine proteinaseBm8172a :: Bm1_06185 - Papain family cysteine protease containing protein partialBm12797c :: Bm1_06175 - cathepsin L-like cysteine proteinaseBm12797b :: Bm1_06175 - cathepsin L-like cysteine proteinaseBm12797a :: Bm1_06175 - cathepsin L-like cysteine proteinaseBm748 :: Bm1_23315 - cathepsin L-like cysteine proteinaseBm8207 :: Bm1_23315 - cathepsin L-like cysteine proteinase
Bm951 :: Bm1_33735 - papain family cysteine proteaseBm6970 :: Bm1_33735 - Cathepsin B group
Bm6197 :: Bm1_34510 - Calpain family cysteine protease
Figure 4
A B
Red: cysteine proteases detected in moltingBlue: cysteine proteases detected in embryonic stages and microfilariaeViolet: cysteine proteases detected across all stages* : cysteine proteases detected only as transcripts
L3 3Hrs
24H
rs
5Day
3HrA
sc
24H
rAsc
48H
rAsc
Mol
ting
L4 AMSo
mAF
Som
MFS
om
L3So
m
UTM
F
EE_R
NAs
eq
IM_R
NAs
eqM
M_R
NAs
eq
L3_R
NAs
eq
L4_R
NAs
eqAM
_RN
Aseq
AF_R
NAs
eq
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The copyright holder for this preprint (whichthis version posted May 6, 2021. ; https://doi.org/10.1101/2021.05.06.439182doi: bioRxiv preprint
Figure 5
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Figure 6
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Figure 7
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The copyright holder for this preprint (whichthis version posted May 6, 2021. ; https://doi.org/10.1101/2021.05.06.439182doi: bioRxiv preprint
A
Figure S1
tSNE - 1
tSN
E-2
B
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-8
-6
-4
-2
0
2
4
6
8
Sem
antic
Spa
ce -
Y
arylesterase activity
calmodulin binding
calmodulin-dependent protein kinase activity
carboxylic ester hydrolase activity
coenzyme binding
electron carrier activity
hydrogen ion transmembrane transporter activity
NADH dehydrogenase (ubiquinone) activity
oxidoreductase activity
oxidoreductase activity (multiple)
structural constituent of ribosome
structural molecule activity
-8 -6 -4 -2 0 2 4 6Semantic Space - X
Log10_pvalue-11.37-10.31-9.26-8.21-7.16-6.11-5.05-4.00-2.95
-10
-8
-6
-4
-2
0
2
4
6
8
Sem
antic
Spa
ce -
Y
antioxidant activity
calcium ion binding
catalytic activity
cobalamin binding
glucosyltransferase activity
GTP binding
GTPase activity
hydrolase activity
hydrolase activity, acting on acid anhydrides
nucleoside-triphosphatase activity
oxidoreductase activity
peptidase activity
purine nucleoside binding
RNA helicase activity
serine-type exopeptidase activitystructural molecule activity
UDP-glucosyltransferase activity
unfolded protein binding
-5 0 5Semantic Space - X
Log10_pvalue-7.252-6.623-5.994-5.365-4.736-4.107-3.478-2.849-2.220
-5
0
5
Sem
antic
Spa
ce -
Y
antioxidant activity
carbohydrate kinase activitycatalytic activity
GTP binding
GTPase activityguanyl nucleotide binding
hydrolase activity, acting on acid anhydrides
nucleoside-triphosphatase activity
oxidoreductase activity
phosphofructokinase activity
protein disulfide oxidoreductase activity
purine ribonucleoside binding
unfolded protein binding
-8 -6 -4 -2 0 2 4 6 8Semantic Space - X
Log10_pvalue-9.638-8.902-8.165-7.428-6.692-5.955-5.218-4.481-3.745
-8
-6
-4
-2
0
2
4
6
8
Sem
antic
Spa
ce -
Yaminoacyl-tRNA editing activity
aminoacyl-tRNA ligase activity
arylesterase activity
carboxylic ester hydrolase activity
electron carrier activity
heme-copper terminal oxidase activity
hydrogen ion transmembrane transporter activity
oxidoreductase activity
oxidoreductase activity, acting on a heme group of donors
oxidoreductase activity, acting on NAD(P)H
ribose-5-phosphate isomerase activity
structural constituent of ribosome
structural molecule activitythreonine-type endopeptidase activity
threonine-type peptidase activity
-5 0 5Semantic Space - X
Log10_pvalue-13.00-11.72-10.45-9.17-7.90-6.62-5.35-4.07-2.80
-5
0
5
Sem
antic
Spa
ce -
Y
acireductone dioxygenase [iron(II)-requiring] activity
acyl-phosphate glycerol-3-phosphate acyltransferase activity
aminoacyl-tRNA hydrolase activity
ATPase activator activity
calcium ion binding
enzyme inhibitor activity
glutamate dehydrogenase [NAD(P)+] activity
holocytochrome-c synthase activity
hydrogen ion transmembrane transporter activity
intramolecular oxidoreductase activity
ribose-5-phosphate isomerase activity
structural constituent of ribosome
structural molecule activity
ubiquinol-cytochrome-c reductase activity
-6 -4 -2 0 2 4 6Semantic Space - X
Oxidoreductase activity
Log10_pvalue-3.523-3.264-3.006-2.747-2.488-2.230-1.971-1.712-1.454
-10
-8
-6
-4
-2
0
2
4
6
8
Sem
antic
Spa
ce -
Y
3-beta-hydroxy-delta5-steroid dehydrogenase activity
antioxidant activity
GTP binding
GTPase activity
guanyl nucleotide binding
hydrolase activity, acting on acid anhydrides
oligosaccharyl transferase activity
oxidoreductase activity
oxidoreductase activity, acting on a sulfur group of donors
protein disulfide oxidoreductase activity
purine ribonucleoside binding
steroid dehydrogenase activity
structural constituent of cuticle
structural molecule activity
-8 -6 -4 -2 0 2 4 6 8Semantic Space - X
Log10_pvalue-11.68-10.83-9.99-9.14-8.30-7.45-6.61-5.77-4.92
Early <<->> Middle Early <<->> Late Middle <<->> Late
GO (M
olec
ular
Fun
ctio
n)
Sign
ifica
ntly
UP
GO (
Mol
ecul
ar F
unct
ion)
Si
gnifi
cant
ly D
OW
N
Size: Frequency of the GO term; Color: log 10 of p-value (FDR)
Figure S2
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Figure S3
5Days–L4L3-24Hrs 5Days– L4 L3- 24Hrs5Days– L4 L3- 24Hrs
5Days Asc - Phase
A B
C
D
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Figure S4
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Figure S5: Wolbachial proteins
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0 2000 4000 6000 8000 10000 12000 140000
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BmL3
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ess
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ess
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ess
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ies
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ies
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ess
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ies
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ies
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ess
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Molting
Sample size
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ies
richn
ess
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BmL4
Sample size
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ies
richn
ess
0 2000 4000 6000 8000 100000
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ies
richn
ess BmL3
3Hrs
24Hrs
5Days
3HrsAsc
24HrsAsc
48HrsAsc
MoltingBmL4
Figure S6: Rarefaction curves
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