In the WOS database, the core collection was chosen to extract relevant information. With “horseshoe crab” as the theme of the search, 1,248 results were found on July 26, 2019. The time range was from 1989 to July 26, 2019. The bibliometric plug-in in WOS was used to count the number of the publications of authors, organizations, journals, countries, or regions. All the information of relevant papers (e.g., title, authors, affiliation, address, and keywords) was selected and exported in a tab-delimited format to construct bibliometric maps by using VOSviewer (Version 1.6.11). Bibliometric tools in WOS were also used to extract other relevant information (the number of publications of each author, country, region, organization, field, and journal; the total number of times the publications were cited; and the proportion of all publications).

The bibliometric plug-in in WOS can list the top 20 authors on the basis of the number of publications. Given that WOS may omit relevant information, relevant data were also collected from PubMed and Google Scholar. Two approaches were used to search relevant publications in Google Scholar. The method for collecting publications that included the word “horseshoe crab” only in titles was designated as G1, and the method for collecting publications that included the word “horseshoe crab” in whole articles was designated as G2. The numbers of publications on horseshoe crabs by the top 20 authors were searched in Google Scholar via G1 and G2. In PubMed, the number of publications on horseshoe crab by these 20 authors over the last 30 years was collected.

VOSviewer 1.6.11 was used to generate bibliometric maps. VOSviewer is a free computer program (available in http://www.vosviewer.com/) used primarily for bibliometric visual analysis (van Eck and Waltman, 2010; Vezyridis and Timmons, 2016; Sweileh, 2018). The bibliometric maps generated by VOSviewer, which is supported by the VOS mapping technique, perform well (Van Eck and Waltman, 2007). The VOSviewer can be used to analyze cooperative relationships within a research field and for hot keyword analysis and clustering analysis (van Eck and Waltman, 2017).

The counting method is full counting, which means each link has the same weight. The maximum number of lines is 1,000, and the minimum strength of lines is zero.

The type of analysis was set as coauthored publication and the units of analysis were set as authors, organizations, and countries (regions included) to visualize the research cooperation in this field. Studies that “published by a large number of authors (country or organization)” were not selected. The following thresholds were set: the minimum number of documents of an author (country or organization) was five, and the minimum number of citations of an author (country or organization) was zero. Thus, in this research, the degree of cooperation was defined as the extent of the coauthorship of publications.

“Co-occurrence” and “all keywords” were chosen to identify the hot keywords of papers on horseshoe crabs. The threshold or the minimum number of occurrences of a keyword was five.

The annual number of publications on horseshoe crabs in Google Scholar (using method G2) increased from 1989 to 2014 (Figure 1A and Table 1). Although a declining trend has been shown since 2015, the annual number of publications on horseshoe crabs in Google Scholar (using method G2) is higher than that of publications on other topics.

In the WOS database, the number of total publications about horseshoe crabs fluctuates roughly (Figure 1B and Table 1). Before 1995, few publications on horseshoe crabs were available. In 1995, the proportion of all publications about horseshoe crabs increased sharply. Afterward, the number fluctuated.

The annual number of publications about horseshoe crabs in PubMed decreased. In PubMed, the highest number of publications about horseshoe crabs was recorded in 1994. In Figure 1B, the annual number of publications about horseshoe crabs from 1989 to 1995 in Google Scholar (using method G1) ranged from 20 to 26. It then fluctuated after 1995 and showed a similar trend to WOS.

The phrase “horseshoe crab” had the highest frequency because it was the name of the study object (Figure 5). In 2000, hot terms were population, beach, photosensors, muscle, and immunodeficiency-virus activity. These results indicated that researchers focused on the vision, immune function, and population distribution of horseshoe crabs during this period. In 2005, the main hot key words were hemocytes, Drosophila, vision, hemolymph, cDNA cloning, specificity, signal transduction, Tridentatus, and Limulus. During this period, researchers emphasized the immune ability and vision of horseshoe crabs and began to study the molecular biology of this species. Hot keywords that appeared in 2010 were horseshoe crab, Limulus-polyphemus, antimicrobial peptides, evolution, growth, temperature, eggs, shorebirds, Arthropoda, lectin, bacterial lipopolysaccharides, and behaviors. In 2015, the hot key words were heavy metals, black tiger shrimp, seasonal movements, phylogeny, arthropod trackways, genomic organization, and Xiphosurida. In recent years, scholars focused on the physiology, phylogeny, and behavior of horseshoe crabs.

We could infer that studies on horseshoe crab have mainly focused on the biochemical reaction, immune ability, medical application, ecology, and conservation of this animal. Biochemistry molecular biology is the hottest field (Table 6). Marine freshwater biology, zoology, environmental sciences ecology, and immunology have also been highly explored.

Google Scholar (using G2 method) might include articles that mentioned horseshoe crabs but did not focus on them, resulting in much more publications found in Google Scholar G2 than in WOS, PubMed, and Google Scholar G1. For example, through G2 searching, studies about the exoskeleton structure (Raabe et al., 2005) and lectin (Moura et al., 2006) were included, but these studies were not about horseshoe crabs because “horseshoe crabs” in these articles were used as references but did not examine horseshoe crabs. The data showed that horseshoe crabs have been widely explored (Figure 1A), and the trend of the number of publications in PubMed is different from that in WOS and Google Scholar (Figure 1B). The number of horseshoe crab publications from 1989 to 1997 in the PubMed database was higher than that in Google Scholar and WOS databases. Google Scholar and WOS database had a high number of horseshoe crab publications. One possible reason was the selective collection of the PubMed database, which tends to focus on the medical field. From 1989 to 1997, medically relevant studies were published and included; from 2003 to 2019, some medically irrelevant horseshoe crab publications (e.g., conservation and basic physiology researches) were excluded by PubMed. According to WOS, the top 20 pioneers have provided many important studies (Table 4). Thus, new learners who are interested in horseshoe crabs are suggested to read the articles of these pioneers. As shown in Table 4, Google Scholar can provide scholars with a large number of publications.

The large number of publications may indicate efforts in research, whereas a high number of citations per article may indicate that these studies are hot spots and attract considerable attention. According to the results, the USA and Japan finished numerous works in horseshoe crab studies (with the first and second-highest number of publications). Publications from France, Japan, Canada, and Singapore have attracted the attention of researchers (a high number of times each article was cited). Kyushu University and the National University of Singapore have released a large number of publications, which may attract more attention from researchers. Articles in the Journal of Biological Chemistry, Developmental and Comparative Immunology, Fish Shellfish Immunology, and Biochemistry have been widely explored. Publications from these countries, organizations, and journals can be helpful as references for people who want to conduct further research on horseshoe crabs.

Cooperation among countries or regions has improved since 1989. However, many authors and organizations rarely cooperate with others. The USA is the most active in cooperation. Kyushu University, the University of Florida, and Rutgers State University may be cooperation cores. Kawabata, Iwanaga, Watson, and Shin often collaborated with others.

Global cooperation was connected by four rounds of the International Workshop on the Science and Conservation of Horseshoe Crabs, which has been held by the Horseshoe Crab Specialist Group of the International Union for Conservation of Nature (IUCN) every 4 years since 2007. As a result, global cooperation and communication have greatly improved in this field. The first International Symposium on the Science and Conservation of Horseshoe Crabs was held in New York, and proceeding papers were collected in the book “Biology and Conservation of Horseshoe Crabs,” which was edited by Tanacredi, Botton, and Smith (Tanacredi et al., 2009). The second, third, and fourth international workshops on the Science and Conservation of Asian Horseshoe Crabs were held in Hongkong, Nagasaki, and Guangxi, respectively. The change in conference subject indicated that researchers were encouraged to focus on Asian horseshoe crabs. The book “Changing Global Perspectives on Horseshoe Crab Biology, Conservation, and Management” edited by Carmichael, Botton, Shin, and Cheung reported a significant progress on scientific research on horseshoe crabs within the past 10 years and collected proceeding papers from the second and third international conferences (Carmichael et al., 2015). As cochairs of the Horseshoe Crab Specialist Group of the IUCN, Botton and Shin provided considerable contributions to this event and the editing of conference proceedings, which accelerated global collaboration on horseshoe crabs.

The hot key words and fields indicated that scholars focused on physiology, medical value, and ecological conservation. Before and in 2000, researchers focused on the vision, immune function, and population distribution of horseshoe crabs. In 2010, researchers continued to focus on the immune ability and vision of horseshoe crabs and began to study the molecular biology of this animal. Scholars focused on the physiology and behavior of horseshoe crabs, and the effects of environmental stress on the horseshoe crabs were also investigated. The trend of variations in research focus showed that horseshoe crab physiology, especially the immune system, has been most widely explored, followed by medical application and conservation.

Horseshoe crab is unique with a strong immune system and famous for the utilization of their hemolymph in endotoxin detection (Fennrich et al., 2016). Over the past 30 years, studies have been conducted to identify the mechanism of this strong immunity. Iwanaga and Kawabata discovered the molecular basis for the innate immunity of horseshoe crab and attributed it to the application of horseshoe crabs' inner defense molecules in medicine (Kawabata et al., 1996; Iwanaga et al., 1998; Beisel et al., 1999; Iwanaga, 2002). Ding and Ho explored the hemolymph protein of horseshoe crabs and the mechanism of immune molecules, which greatly contributed to bacterial pyrogen testing (Tan et al., 2000a,b; Ding and Ho, 2001; Frecer et al., 2004; Zhu et al., 2004; Ng et al., 2007; Yu et al., 2009). Armstrong mainly focused on alpha (2)-macroglobulin and found that it is important for horseshoe crab hemolysis and immune system because of its special function (Armstrong et al., 1998; Armstrong and Quigley, 1999). The high citation frequency of these authors' works proved the high degree of concern for the immune system of horseshoe crabs (Table 4).

Given the ability of amebocytes to instantaneously react with endotoxins, horseshoe crabs are hunted for use in safety tests in medical production (Gauvry, 2015; Fennrich et al., 2016; Krisfalusi-Gannon et al., 2018). Tachyplesin in horseshoe crab hemocytes exerts a strong antimicrobial effect on Gram-positive and Gram-negative bacteria (Dorrington and Gomez-Chiarri, 2008) and has an antimicrobial activity against multidrug-resistant pathogens (Liu et al., 2018). Tachyplesin kills bacteria by targeting FabG, which is the conserved β-ketoacyl-acyl carrier protein reductase in unsaturated fatty acid biosynthesis (Liu et al., 2018). However, tachyplesin may target homologous enzymes in mammalian cells, causing cytotoxicity (Liu et al., 2018). In view of the mechanism of tachyplesin, more researchers should focus on the discrimination between bacterial and mammalian FabG proteins to select specific inhibitory ligands and develop safe antibiotics (Liu et al., 2018). Tachyplesin I from horseshoe crabs shows an antitumor activity; however, the action mode of tachyplesin I in tumor cells remains unclear (Li et al., 2017). The new antimicrobial peptide polyphemusin III can induce cell death by destroying the plasma membrane (Marggraf et al., 2018). Purified recombinant Factor C may be applied to inhibit inflammation and septic shock (Li et al., 2007). The 18-mer peptide T22 in horseshoe crab can target cells quickly through a receptor-specific endosomal route, showing a promising capacity for intracellular delivery (Unzueta et al., 2012). CrSPI, a serine protease inhibitor in horseshoe crabs, can modulate immune responses, including complements and PPO-mediated antimicrobial activities (Jiang et al., 2009). The outer shells of horseshoe crabs can be utilized as burn dressings to accelerate wound healing (Kumar et al., 2016).

The population of horseshoe crabs has declined because of marine environment changes and overhunting for feed and medical use (Tanacredi and Portilla, 2015; Krisfalusi-Gannon et al., 2018). Their spawning grounds and habitat have degraded (John et al., 2018). High acid volatile sulfide values contribute to a decline in the horseshoe crab population in Yamaguchi Bay, Japan (Moqsud et al., 2017). In 2015, 583,000 horseshoe crabs were harvested as eel and whelk bait, and 559,903 horseshoe crabs were harvested for biomedical production (Krisfalusi-Gannon et al., 2018). Although horseshoe crabs are released after medical production or studies, they would become vulnerable, and their mortality rate might increase (Walls and Berkson, 2003; Leschen and Correia, 2010). Female horseshoe crabs released back to the sea encounter difficulties in spawning (Anderson and Chabot, 2013). A green alga of the family Ulvaceae might cause harm to horseshoe crabs in their natural habitats (Braverman et al., 2012). Moreover, population decline would affect other organisms in food webs involving horseshoe crabs. Horseshoe crabs and their eggs are the food of many animals, such as shorebirds (Berkson and Shuster, 1999; Smith and Berkson, 2005), American eel, and sand shrimp (Perry, 1931; Price, 1962; Botton and Haskin, 1984; Botton and Ropes, 1989; Botton, 2009). Adult horseshoe crabs feed on bivalves (Botton and Ropes, 1989). Therefore, protecting horseshoe crabs is a matter of concern. Using recombinant Factor C as a synthetic alternative to horseshoe crab blood may be a partial solution for horseshoe crab conservation (Maloney et al., 2018), and placing limitations on the exploitation of this animal and the reasonable management of their habitats are keys to conservation.

Global marine environmental problems, such as ocean acidification and warming, drew increased attention in the twenty-first century. Thus, many studies have focused on the effect of these environmental stresses on marine organisms. As shown in Figure 5, in 2010, researchers began to focus on the effects of the changes in temperature on horseshoe crabs and the advantage of horseshoe crab biological adaptation to environmental stress. Botton et al. contributed valuable research on the distribution and developmental ecology of L. polyphemus (Botton and Loveland, 2003; Botton et al., 2003, 2006, 2010; Botton, 2009) and determined the effect of heavy metals, such as copper and cadmium, on horseshoe crab embryos and larvae (Botton et al., 1998; Itow et al., 1998; Botton, 2000). Shin and Cheung cooperated on many studies on horseshoe crab aquaculture and ecology (Hu et al., 2009, 2013; Shin et al., 2009; Kwan et al., 2015, 2016, 2017). They further explored the effect of stressful habitat conditions, such as starvation and hypoxic exposure, on horseshoe crabs (Hu et al., 2010, 2011; Shin et al., 2014). Studies have provided valuable chromosome-level genome or transcriptome data on horseshoe crab (Gong et al., 2019; Liao et al., 2019b) and may be useful for horseshoe crab research.

A large number of studies in various fields have focused on horseshoe crabs. However, research progress on horseshoe crabs is not so rapid for some reasons. First, more cooperation should be encouraged. Our analysis revealed that authors, countries, regions, and organizations mostly become involved in cooperation (Tables 2–4 (indicating more publications), e.g., the USA and Japan, Kyushu University, and Rutgers State University, Kawabata, and Iwanaga. Cooperation would accelerate and contribute to research on horseshoe crabs by providing more studies. Second, wild horseshoe crabs are difficult to obtain. The habitats of horseshoe crabs are not global and only include a few areas; in addition, the horseshoe crab population is declining (Adibah et al., 2012; Liao et al., 2019a). Captive breeding and artificial rearing should be explored, and some researchers searched for good sperm donors (Sheikh et al., 2019). Lastly, conducting research on horseshoe crabs in labs may be difficult. Time would decelerate our research because of the life span of horseshoe crabs. Horseshoe crabs take 10 years to sexually mature (Smith and Berkson, 2005). Mortality is high during the development of juvenile horseshoe crabs into adults (Gauvry, 2015). Relevant studies have shown that growth and survival decrease over 128 days regardless of diet composition (Carmichael et al., 2009). The survival rate of horseshoe crabs in labs may vary in practice. The hemolymph of horseshoe crabs can clot easily. The addition of LPS-free reagents at low and consistent temperatures may prevent clotting (Armstrong and Conrad, 2008). In the future, attempts to solve these problems are worth making.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.