T. canis is a zoonotic roundworm found worldwide, capable of causing severe clinical manifestations such as blindness and neurological disorders, especially in pediatric and adolescent populations from impoverished communities [27, 28]. The advent of high throughput sequencing technique, coupled with advanced bioinformatics, has significantly facilitates the research of T. canis, an economically significant parasite [29]. In 2015, we sequenced and annotated the draft genome of T. canis, revealing at least 18,596 protein coding genes, including 355 TFs and 870 excretory-secretory proteins (ESPs) with various functions [10]. Over the past few decades, mass spectrometry (MS)-based proteomics has become crucial for elucidating the dynamic and diverse proteomic profiles of cells and tissues, despite limitations in capturing the entire proteome. In the last decade, improvements in sample preparation, peptide separations, data processing, and the development of faster and more sensitive MS instruments have pushed the boundaries of depth and throughput in proteomics [30]. Now, one-hour human proteome has reached by using the new Orbitrap Astral platform, ushering unprecedented studies of biological impact by harmony of throughput, depth and sensitivity [31]. In this study, we identified 8565 proteins from adult T. canis using the Orbitrap Astral platform, which is a substantial increase compared to the 582 somatic proteins identified from the L3 larvae by the Q Exactive Orbitrap platform in a previous study [12]. Comparing with the previously published data revealed that 507 proteins were identified in both L3 larvae and adult T. canis, and 75 proteins were uniquely identified from the L3 larvae, including some C-type lectins (CTLs) (e.g., TES-32 and collectin-12) (Additional file 1: Table S1). It is hypothesized that Toxocara parasites possibly evade host immune response by secreting abundant functional CTLs [32]; among which, TES-32 is a major component of ESPs secreted by T. canis larvae [33, 34]. These findings suggested that the strategies adopted by T. canis for survival or development in the host may be different between its larval stage and adult stage. In addition, acetyl-coenzyme A synthetase and pyruvate dehydrogenase complex were identified in the larvae, whereas only pyruvate dehydrogenase complex was identified in adult T. canis, suggesting that T. canis may have stage-specific differences in energy utilization mechanisms. Further research on stage-specific proteins expression will enhance our understanding of the migration and development processes of T. canis in its hosts.

The protein kinases are a very extensive proteins family that share a conserved catalytic core common with both serine/threonine and tyrosine protein kinases, which play essential roles in a wide range of cellular processes, including division, differentiation, proliferation, apoptosis, and are responsible for phosphorylation [35]. Our previous genomic analysis predicted the presence of 458 protein kinases in T. canis genome [10]. In this study, we further identified 188 proteins with “protein kinase domain” at the protein level, including over 30 serine/threonine proteins. Moreover, GO annotation revealed that 253 proteins were enriched in “protein kinase activity” term under the “cellular component” category, and 258 proteins were enriched in “protein phosphorylation” term under the “biological process” category (Fig. 2b), suggesting that these protein kinases may play an important role in T. canis. Another significant protein group is the “major sperm protein (MSP) domain” proteins, unique to nematodes, which are essential for the amoeboid motility of nematode sperm, independent of actin [36]. In this study, we identified 67 proteins with “MSP domain” at the protein level, which may be the potential candidate proteins to control T. canis reproduction.

TFs are key regulators of gene expression, modulating the transcriptional activation or repression of target genes [37]. Our previous genomic study of T. canis revealed that T. canis encoded 355 TFs [10]. In this study, we identified 93 T. canis TFs belonging to 28 families. There are at least 42 zinc finger TFs in T. canis, including 27 ZBTB family TFs, 6 zf-C2H2 family TFs and 2 zf-GATA family TFs. By comparing our result with the previous study [12], we found that A0A0B2URD4 [3-methyl-2-oxobutanoate dehydrogenase (2-methylpropanoyl-transferring)], one of the CCAAT/enhancer binding protein (C/EBP) TFs family, can be identified from both L3 larvae and adult T. canis. By comparing with female adult T. canis, the expression level of A0A0B2URD4 was up-regulated 2.14-folds in male adult T. canis. The C/EBP TFs play crucial roles in regulating numerous significant biological processes, such as inflammation, cell proliferation and differentiation, energy metabolism, and signal transduction [38]. Further investigation into the biological functions of TFs, particularly those with stage- and gender-specific expressed TFs, will help to better reveal the development processes, spermatogenesis or egg generation of T. canis.

The genome data predicted that T. canis would produce at least 870 ESPs [10], and the proteome analyses identified 79 ESPs of T. canis L3 larvae [12, 29, 39]. In this study, we identified 385 “extracell protein”, seven of which correspond to previously identified ESPs from TES of L3 larvae, including A0A0B2V6Q8 (paramyosin), A0A0B2V815 (macrophage migration inhibitory factor-like protein, or l-dopachrome isomerase), A0A0B2W1X6 (transthyretin-like protein 46), A0A0B2UNP1 (laminin subunit beta-1), A0A0B2W1F7 (laminin-like protein lam-2), A0A0B2VHM0 (apolipophorin) and A0A0B2V9X0 (galectin). It is generally recognized that proteins consistently expressed throughout different stages of the life cycle may serve as effective vaccine candidate, although this idea is still speculative, given the relatively limited research in helminth immunology and vaccine development compared to other medical fields. Moreover, we found that none of these seven proteins identified from both L3 larvae TES and adult T. canis was PDAs in this study, suggesting that they are not gender-specific proteins and may play important roles in the process of T. canis migration and development, and these seven ESPs may be promising vaccine candidates for the prevention of toxocariasis [40,41,42]. Besides, some “cell membrane protein” (n = 429) are also important vaccine candidates of toxocariasis, such as A0A0B2VEA6 (enolase) and A0A183UM88 (cathepsin L), which need to be further investigated [43,44,45].

Compared to male adult T. canis, 682 up-regulated PDAs and 844 down-regulated PDAs were identified in female adult T. canis, including 139 proteins exclusively expressed in female T. canis and 272 proteins exclusively expressed in male T. canis in this study. It should be noted that during the mating process of female and male adult T. canis, the sperms of the male adult enter the body of the female, leading to the detection of male-specific expressed proteins within the female adult, such as some “major sperm protein (MSP) domain” proteins. MSPs constitute 15–20% of the total protein in nematode sperm and are absent from all other cell types in the worm [46]. Although the abundance of these male-specific expressed proteins is generally low in female adults and does not affect the type and quantity of PDAs, it will affect the statistics and analysis of gender-specific expressed proteins. This phenomenon will result in a lower count of male-specific expressed proteins than the actual situation. Previous study has shown a higher number of male-specific genes compared to female-specific genes, with 321 genes exclusively transcribed in female T. canis and 1467 genes exclusively transcribed in male T. canis [11]. Vaccination is still the most effective strategy for combating infectious diseases; however, the development of vaccines against multicellular helminths is riddled with obstacles due to its huge size, and constantly migration through various tissues under multiple life cycle stages [47]. To date, there are still no any reliable vaccine against zoonotic soil-transmitted helminths (STHs) [48]. Analysis and research of the PDAs between female and male adult T. canis, especially those gender-specific proteins related to spermiogenesis and egg generation, may facilitate the discovery of new vaccine candidates, such as major sperm proteins, chondroitin proteoglycan 1 and chitin-binding type-2 domain-containing protein. Many up-regulated PDAs, such as chondroitin proteoglycan 1, chondroitin proteoglycan 2 and chitin-binding type-2 domain-containing protein with “chitin binding domain”, were enriched in the GO terms of “aminoglycan metabolic process”, “chitin metabolic process” and “chitin binding”, indicating that they could contribute to the formation of the T. canis eggs in female adult T. canis.

Our previous study of the T. canis genome predicted that T. canis would produce 408 phosphatases [10]. In this study, we have also identified many phosphatases at the protein level. The protein tyrosine phosphorylation (PTP) is a prevalent post-translational modification that influences protein stability and regulates enzyme activity. Thus, maintaining an appropriate level of PTP is crucial for various cellular functions, including cell growth, proliferation, differentiation, and transformation, with particular importance during sperm capacitation [49, 50]. The PTP stands out as a critical intracellular signaling event that regulates sperm function, and the capacitated sperms show high levels of PTP, so sperm PTP is a meaningful indicator of capacitation [49, 51]. In the present study, many down-regulated PDAs, such as protein-tyrosine phosphatase, receptor-type tyrosine-protein phosphatase S and tyrosine-protein phosphatase non-receptor type 14 with “Protein-tyrosine phosphatase, receptor/non-receptor type” domain, were enriched in the GO terms of “protein tyrosine phosphatase activity”, suggesting that these proteins could play important roles in spermatogenesis and capacitation of T. canis.

In this study, we identified 21 TFs with differential abundance, belonging to 10 families, with 10 up-regulated PDAs and 11 down-regulated PDAs between female and male adult T. canis, as well as 3 TFs (e.g. A0A0B2VQ53, A0A183UMK5 and A0A183V3J6) exclusively expressed in female T. canis and 3 TFs (e.g. A0A0B2V842, A0A183VAA5 and A0A183V625) exclusively expressed in male T. canis. The roles of differentially expressed TFs between female and male adult T. canis may be critically important, particularly those that are sex-specific. In female T. canis, these TFs may potentially regulate genes involved in ovarian development and oocyte maturation, while in males, they may regulate genes associated with testicular development and spermatogenesis. These differentially expressed TFs may shape the biological characteristics of sexual dimorphism by regulating sex-specific gene expression, influencing reproductive system development, and modulating behavior and adaptability. However, research on the 21 differentially expressed TFs identified in this study remains limited to date. T. canis of different genders exhibit distinct physiological states and therefore may have different ESPs. In this study, we predicted the “extracell protein” by subcellular localization to explore the potential role of these PDAs. A total of 69 “extracell protein” were identified, including 46 up-regulated PDAs and 23 down-regulated PDAs, as well as 6 proteins specifically expressed in female adult T. canis (e.g. cysteine-rich motor neuron 1 protein) and 12 proteins specifically expressed in male adult T. canis (e.g. ephrin type-A receptor 3, ancylostoma secreted protein, orcokinin peptides type B and EGF_CA domain-containing protein). Further investigation of these proteins will enhance our understanding of the biological function of T. canis.

Helminths exhibit a sophisticated life cycle that includes various stages within the same host, with distinct antigen expression as they migrate and develop. They have developed mechanisms to modulate the host's immune responses, enabling them to escape immunological attack. By evaluating the efficacy of complex mixtures of parasitic lysates and secretions, and the individual proteins against STHs infection, researchers are becoming aware that there are still many difficulties and challenges in the vaccine development of STHs [48], for example, the lack of effective targets, high-quality genome data and comprehensive protein annotation information. Although 8565 T. canis proteins were identified based on T. canis protein database downloaded from UniProtKB (released on March 6, 2024) in this study, it should be emphasized that the information of a few proteins is still constantly being updated and changed in UniProtKB database, such as A0A0B2V815. In a previous study, A0A0B2V815 is called macrophage migration inhibitory factor-like protein based on the UniProtKB database [12]; however, it is called L-dopachrome isomerase in the current UniProtKB database (updated on January 24, 2024). As one of the major STHs and zoonotic parasite threatening animal and human health worldwide, the fundamental studies on molecular biology and pathogenesis of T. canis still need to be further strengthened to break through the bottlenecks in diagnostic methods and vaccine development.

Despite the comprehensive proteomic analysis conducted in this study, some limitations should be acknowledged. First, while the use of the Orbitrap Astral platform allowed for the identification of a substantial number of proteins in T. canis, the complexity and diversity of the proteome suggest that it is unlikely that the entire spectrum of expressed proteins was captured, particularly those with low abundance or transient expression. Second, while differential protein expression was identified between male and female adult T. canis, the functions of these proteins, especially TFs and ESPs, warrant further experimental validation.