Insights into the L3 to L4 developmental program through ...May 06, 2021 · during molting (‘Molting’; Days 8/9) and finally as viable L4 larvae (‘BmL4’). The animal procedures

Brugia malayi L3 to L4 development

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Insights into the L3 to L4 developmental program through proteomics 1

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Sasisekhar Bennurua, Zhaojing Mengb, James McKerrowc, Sara Lustigmand and 3

Thomas B Nutmana 4

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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

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*Running title: Brugia malayi L3 to L4 development 14

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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

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 was not certified by peer review) is the author/funder.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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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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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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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



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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



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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



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(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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19

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



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20

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specific proteomic profiling. PLoS neglected tropical diseases 3:e410. 575

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Acad Sci U S A 102:15545-50. 579

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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



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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



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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



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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



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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



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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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B C

Figure 1

A

BmL3

3Hr

24Hr

24HrAsc

3HrAsc

5Days

48HrAsc

Molting

BmL4



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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



https://doi.org/10.1101/2021.05.06.439182

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





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



https://doi.org/10.1101/2021.05.06.439182

Figure 5



https://doi.org/10.1101/2021.05.06.439182

Figure 6



https://doi.org/10.1101/2021.05.06.439182

Figure 7



https://doi.org/10.1101/2021.05.06.439182

A

Figure S1

tSNE - 1

tSN

E-2

B



https://doi.org/10.1101/2021.05.06.439182

-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


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


carbohydrate kinase activitycatalytic activity

GTP binding

GTPase activityguanyl nucleotide binding


nucleoside-triphosphatase 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



oxidoreductase activity, acting on a heme group of donors

oxidoreductase activity, acting on NAD(P)H

ribose-5-phosphate isomerase activity


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


intramolecular oxidoreductase activity

ribose-5-phosphate isomerase 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


GTP binding

GTPase activity

guanyl nucleotide binding


oligosaccharyl transferase activity


oxidoreductase activity, acting on a sulfur group of donors

protein disulfide oxidoreductase activity

purine ribonucleoside binding

steroid dehydrogenase activity

structural constituent of cuticle


-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



https://doi.org/10.1101/2021.05.06.439182

Figure S3

5Days–L4L3-24Hrs 5Days– L4 L3- 24Hrs5Days– L4 L3- 24Hrs

5Days Asc - Phase

A B

C

D



https://doi.org/10.1101/2021.05.06.439182

Figure S4



https://doi.org/10.1101/2021.05.06.439182

Figure S5: Wolbachial proteins



https://doi.org/10.1101/2021.05.06.439182

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BmL3

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3Hrs

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24Hrs

Sample size

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ies

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Sample size

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ies

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3HrAsc

Sample size

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ies

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ess

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24HrAsc

Sample size

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ies

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ess

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48HrAsc

Sample size

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ies

richn

ess

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Molting

Sample size

Spec

ies

richn

ess

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BmL4

Sample size

Spec

ies

richn

ess

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Sample size

Spec

ies

richn

ess BmL3

3Hrs

24Hrs

5Days

3HrsAsc

24HrsAsc

48HrsAsc

MoltingBmL4

Figure S6: Rarefaction curves



https://doi.org/10.1101/2021.05.06.439182

Documents

Insights into the L3 to L4 developmental program through ...May 06, 2021 · during molting (‘Molting’; Days 8/9) and finally as viable L4 larvae (‘BmL4’). The animal procedures