**1. Introduction**

Sea cucumbers are marine invertebrates that belong to the class Holothuroidea of the phylum Echinodermata. Globally, there are about 1500 species of sea cucumbers [1], which are divided into three subclasses: Aspidochirotacea, Apodacea, and Dendrochirotacea, and can be further divided into six orders: Aspidochirotida, Elasipodida, Apodida, Molpadida, Dendrochirotida, and Dactylochirotida [2].

Sea cucumbers are found in benthic areas and the deep sea worldwide [3]. They play an important role in marine ecosystems and occupy a similar niche to earthworms in terrestrial ecosystems [4]. Sea cucumbers obtain food by ingesting marine sediments or filtering seawater [5] and provide a unique, fertile habitat for a variety of microorganisms, including bacteria and fungi [6]. However, since most microorganisms are unculturable under conventional laboratory conditions [7], this review primarily focuses on culturable sea-cucumber-associated microorganisms.

Sea cucumbers have been used in medicine in Asia for a long time [8]. For example, an ointment derived from the sea cucumber *Stichopus* sp. 1 is used to treat back and joint pain in Malaysia [9]. Compounds isolated from sea cucumbers have a variety of biological and pharmacological activities, such as anticancer, antiangiogenic, anticoagulant/antithrombotic, antioxidant, antiinflammatory, antimicrobial, antihypertension, and radioprotective properties [10,11]. A phase II clinical trial of a sea cucumber extract, called TBL-12, has been conducted in patients with untreated asymptomatic myeloma [12]. Many studies have shown that the microorganisms associated with marine animals, such as sponges and ascidians, are the true producers of marine natural products [13–16]. Therefore, investigating sea-cucumber-associated microorganisms is essential for discovering new compounds with potential as novel active drugs. For the past 20 years, there has been an increasing effort made by researchers on diversity and bioactive compounds of microorganisms associated with sea cucumber. However, previously, no comprehensive review article as such has ever been published about this field.

This review discusses the biodiversity of the culturable microorganisms associated with sea cucumbers and the chemical structure and bioactive properties of the secondary metabolites produced by these microorganisms.

#### **2. Microorganisms Associated with Sea Cucumbers**

#### *2.1. Geographical Distribution of Microorganisms Associated with Sea Cucumbers*

Although sea cucumbers are distributed in oceans worldwide [3], most studies on the biological and chemical diversity of sea-cucumber-associated microorganisms have focused on species in the northern temperate areas and tropical areas of the eastern hemisphere [17–26]. More than 80% of the sampling sites are located on the west coast of the Pacific Ocean. However, a small number of sampling sites are also located in the Atlantic, Indian, and Antarctic Oceans [17–26] (Figure 1 and Table S1). Sea cucumber samples are typically collected from the coast at a depth of less than 20 m [17–21].

**Figure 1.** Geographical distribution of sea cucumber samples used for studies of culturable microorganisms. The red circles represent sampling sites: (A) Funka Bay and Ainuma fishing port, Hokkaido, Japan; (B) Sea of Japan, Russia; (C) Yellow Sea, China; (D) Geomun-do, Yeosu, Korea; (E) Kushima, Omura; Koecho; Nagasaki; Japan; (F) Coast of Aka Island, Okinawa prefecture, Japan; (G) Ningde, Fujian, China; (H) South China Sea, China; (I) Dayang Bunting Island, Yan, Kedah Darul Aman, Malaysia; (J) Tioman Island, Pahang Darul Makmur; Peninsular Malaysia; Pangkor Island, Perak; Malaysia; (K) Sari Ringgung, Lampung, Indonesia; (L) Larak Island, Persian Gulf, Iran; (M) Tabarka, Tunisia; and (N) the Antarctic.

#### *2.2. Culturable Microorganisms Associated with Sea Cucumbers*

The sea cucumbers used for the isolation of culturable microorganisms belong to five genera (*Holothuria*, *Cucumaria*, *Stichopus*, *Apostichopus*, and *Eupentacta*) in four families (Holothuriidae, Stichopodidae, Cucumariidae, and Sclerodactylidae) (Table 1). The dominant species is *Apostichopus japonicus*, which accounts for about 41% of the total sea cucumber population. In second place, *Holothuria leucospilota* accounts for about 27% of the total sea cucumber population (Table S1).

In studies on microorganisms associated with sea cucumbers, samples are primarily obtained from the following body parts: the body wall [22,23], body surface [18,21,24–29], inner body tissue [30], coelomic fluid [24,31], stomach [30], intestines [4,6,17,19,25,32–35], brown gastrointestinal tissue [30], and feces [20,22].

Sea cucumbers harbor a rich and diverse assortment of microorganisms. A variety of microorganisms, including bacteria and fungi, have been isolated from sea cucumbers. Most of the isolation conditions (medium, temperature, and aeration) are common. There are some papers on the diversity of culturable bacteria associated with sea cucumbers, which plays a very important role in understanding the digestion and diseases of sea cucumbers [4,6,17,25,33]. Because marine-derived fungi had shown potential to synthesize

pharmaceutical compounds with bioactivities, researchers usually directly isolate fungi associated with sea cucumbers for the separation of active natural products [21,28,29], except one paper about the diversity and bioactivity of fungi associated with sea cucumbers [22].
