Fauna of anemonefishes (Pomacentridae,Amphiprioninae) and their host sea anemones (Cnidaria,Actiniaria) on reefs of Phu Quy,Con Son,and An Thoi Islands (South China Sea,South Vietnam,and Gulf of Thailand) and a review of these groups from the coastal waters of Vietnam

A full species composition of anemonefishes (Pomacentridae, Amphiprioninae) and their host sea anemones (Cnidaria, Actiniaria) from reefs of Phu Quy and Con Son Islands (off South Vietnam) and An Thoi Islands (northeastern Gulf of Thailand) is presented for the first time. A comparison of the faunas of anemonefishes and their host sea anemones is conducted in the coastal waters of Vietnam and adjacent waters. The presence of intermittent ranges of the anemonefishes in the coastal waters of Vietnam is demonstrated. For example, the ranges of two species, Amphiprion clarkii and A. frenatus, include the coastal waters of Vietnam, excluding the Gulf of Thailand, where they are not found even at the eastern border of the gulf. A. perideraion is absent in the Gulf of Tonkin, but it is distributed to the south along the entire coastal zone of Vietnam, including the Gulf of Thailand. In the coastal waters of Vietnam, A. sandaracinos is distributed only between the Gulf of Tonkin and Gulf of Thailand. A. polymnus is reliably indicated in the coastal waters of Vietnam between 12°50′ and 9°54′ N, but, most likely, its range is continuous along the entire Vietnamese coast. The richness of species composition of host sea anemones decreases (from nine to three species) in the southern direction, from Central Vietnam to the eastern border of the Gulf of Thailand.     

Searching for a Toxic Key to Unlock the Mystery of Anemonefish and Anemone Symbiosis

Twenty-six species of anemonefish of the genera Amphiprion and monospecific Premnas, use only 10 species of anemones as hosts in the wild (Families: Actiniidae, Stichodactylidae and Thalassianthidae). Of these 10 anemone species some are used by multiple species of anemonefish while others have only a single anemonefish symbiont. Past studies have explored the different patterns of usage between anemonefish species and anemone species; however the evolution of this relationship remains unknown and has been little studied over the past decade. Here we reopen the case, comparing the toxicity of crude venoms obtained from anemones that host anemonefish as a way to investigate why some anemone species are used as a host more than others. Specifically, for each anemone species we investigated acute toxicity using Artemia francisca (LC₅₀), haemolytic toxicity using ovine erythrocytes (EC₅₀) and neurotoxicity using shore crabs (Ozius truncatus). We found that haemolytic and neurotoxic activity varied among host anemone species. Generally anemone species that displayed greater haemolytic activity also displayed high neurotoxic activity and tend to be more toxic on average as indicated by acute lethality analysis. An overall venom toxicity ranking for each anemone species was compared with the number of anemonefish species that are known to associate with each anemone species in the wild. Interestingly, anemones with intermediate toxicity had the highest number of anemonefish associates, whereas anemones with either very low or very high toxicity had the fewest anemonefish associates. These data demonstrate that variation in toxicity among host anemone species may be important in the establishment and maintenance of anemonefish anemone symbiosis.     

Assemblage and interaction structure of the anemonefish-anemone mutualism across the Manado region of Sulawesi,Indonesia

The Manado area (Indonesia–North Sulawesi), a marine high diversity hot-spot, hosts 7 species of anemonefish (family Pomacentridae, subfamily Amphiprioniae) living in symbiosis with 9 species of sea anemones (family Stichodactylidae and Actiniidae). This high biological diversity −27% and 80%, respectively, of the total known diversity of anemonefish and sea anemones—allows us to test different hypotheses focused on the obligate mutualism between anemonefish and sea anemones. In the Manado area, species richness of anemones and anemonefish across several sites was not correlated, but all anemones contained at least one fish individual, and there was a strong positive correlation between the numbers of individual anemonefish and anemones. As expected, each fish species had a preferred anemone host; also a partial niche overlap (Pianka’s Index) was often detected. The analysis of unique species composition suggests that competition is not an important factor determining the presence or absence of particular combinations of either anemonefish or host anemones (no evidence of competitive exclusion). The NODF algorithm showed that, at both a regional and local scale, the interaction between anemonefish and host anemones is not significantly nested, as a result of a combination of local conditions with competition, forcing species that regionally are more generalist to become more specialist.     

Juvenile Thalassoma amblycephalum Bleeker (Labridae, Teleostei) dwelling among the tentacles of sea anemones: A cleanerfish with an unusual client?

At least 51 species of fishes are facultative symbionts of sea anemones. Most of the behavioural, ecological and physiological aspects of these associations including their costs and benefits are unknown. We recorded the behaviour and the habitat use of eight assemblages (three or ten specimens each) of the juvenile wrasse Thalassoma amblycephalum dwelling among the tentacles of the two sea anemones Entacmaea quadricolor (clonal type), and Heteractis magnifica at a coral reef in southern Japan during 16 months in daylight hours. There are only two past records of this facultative association, one from east Africa and one from Indonesia. The wrasse remained close to and was occasionally in physical contact with the host when foraging amongst the tentacles. When frightened, they took shelter among corals, away from the host anemone. The wrasse co-existed with the anemonefishes Amphiprion frenatus in E. quadricolor and A. ocellaris in H. magnifica. By using forced host contact tests ex situ and scanning electron microscopy examination of the fish epidermis, we show that juveniles of this wrasse are protected from E. quadricolor, but possibly not from H. magnifica. We suggest that juvenile T. amblycephalum dwelling among the tentacles of sea anemones are cleanerfish with an unusual client, in that they appear to clean mucus and, or, necrotic tissue from the sea anemone host.     

Coexistence of two anemonefishes,Amphiprion clarkii and A. perideraion,which utilize the same host sea anemone

Synopsis Social structure and interactions between the anemonefishes, Amphiprion clarkii and A. perideraion, which utilize the same host sea anemone Radianthus kuekenthali, were investigated on a coral reef of Okinawa Islands, Japan. In an 87 × 373 m² study area, 98 sea anemones were inhabited by both species (32.5%), by only A. clarkii (48.9%), or by only A. perideraion (18.6%). A group of A. clarkii often occupied two or more individual hosts, and group members often interchanged. However, a group of A. perideraion usually used only one host and migration between groups was rare. The larger A. clarkii suppressed reproduction of A. perideraion in cohabiting groups, while A. perideraion suppressed settlement of Juvenile A. clarkii to its own hosts. Juvenile A. clarkii settled on small hosts as well as on large hosts, whereas juvenile A. perideraion settled only on large hosts. Coexistence appears to be possible in part by differences in settlement patterns between juveniles of the two anemonefishes.     

Factors affecting the local abundance of two anemonefishes (Amphiprion frenatus and A. perideraion) around a semi-closed bay in Puerto Galera,the Philippines

Many marine organisms disperse or migrate among habitats, which affects their abundance patterns at individual local habitats. To clarify the factors affecting the distribution patterns of two anemonefishes (Amphiprion frenatus and A. perideraion), we measured the habitat patch size (anemone size), patch isolation (mean distance from other anemones), presence/absence of other anemonefish species, depth, and abundance of the two anemonefishes at each anemone around a semi-closed bay (up to 3.7 km) in Puerto Galera, the Philippines. We assumed that local abundance increases with habitat size and decreases with patch isolation because of greater resource availability and reduced rates of recruitment from other patches. Local abundance of A. frenatus was related to habitat size and the presence of other anemonefish species, whereas that of A. perideraion was affected by the presence of other anemonefish species and water depth. Interspecific competition and/or niche differentiation of habitat can explain the negative relationship between the local abundance of the target species and other anemonefish. Patch isolation was not significant for both species probably because the dispersal rate was not directly proportional to the geographic distance between patches at our study site.     

Do the Anemonefish Amphiprion ocellaris (Pisces: Pomacentridae) Imprint Themselves to Their Host Sea Anemone Heteractis magnifica (Anthozoa: Actinidae)?

Juvenile anemonefishes detect their host sea anemone by olfactory stimuli; in order to investigate whether this behaviour is innate or acquired, the anemonefish species Amphiprion ocellaris was bred in two different ways: 1. With no host sea anemone present at all (–A); and 2. With the specific host sea anemone Heteractis magnifica present in the hatching aquarium, so that these eggs were laid and hatched close to the sea anemone, as in nature (+A). The two different types of juvenile A. ocellaris were presented to the odours of the host sea anemone H. magnifica in two sets of short-term experiments with the host (a) visually hidden in a net cage, and (b) visible but physically separated from the anemonefishes. In both cases, a water flow was established between fishes and host. The +A-fishes found their host by olfactory and not by visual stimuli. In both series, the –A-fishes showed a significantly lower affinity behaviour towards the odour compounds from the host sea anemone than the +A-fishes did. A third type of experiment was a direct confrontation between fishes and host; here, the –A-fishes were indifferent towards the host sea anemone for almost 48 h, while the +A-fishes acclimated to the host sea anemone within the first 5 min of the direct confrontation. The results of this study suggest that Amphiprion ocellaris imprints itself olfactorily to its species-specific host sea anemone Heteractis magnifica, and, furthermore, may be genetically disposed towards olfactory recognition of the host sea anemone.     

Anemonefish depletion reduces survival,growth, reproduction and fishery productivity of mutualistic anemone–anemonefish colonies

Intimate knowledge of both partners in a mutualism is necessary to understand the ecology and evolution of each partner, and to manage human impacts that asymmetrically affect one of the partners. Although anemonefishes and their host anemones are iconic mutualists and widely sought by ornamental fisheries, the degree to which anemones depend on anemonefishes, and thus the colony-level effects of collecting anemonefishes, is not well understood. We tracked the size and abundance of anemone Entacmaea quadricolor and anemonefish Amphiprion melanopus colonies for 3 yr after none, some, or all of the resident anemonefish were experimentally removed. Total and partial removal of anemonefish had rapid and sustained negative effects on growth, reproduction and survival of anemones, as well as cascading effects on recruitment and productivity of anemonefish in the remaining colony. As predicted, total removal of anemonefish caused acute declines in size and abundance of anemones, although most anemone colonies (76 %) slowly resumed growth and reproduction after the arrival of anemonefish recruits, which subsequently grew and defended the hosts. Partial removal of anemonefish had similar but typically less severe effects on anemones. Remarkably, the colony-level effects on anemones and anemonefish were proportional to the size and number of anemonefish that were experimentally removed. In particular, anemone survival and anemonefish productivity were highest when one or more adult anemonefish remained in the colony, suggesting that adult fish not only enhanced the protection of anemones, but also increased the recruitment and/or survival of conspecifics. We conclude that the relationship between E. quadricolor and A. melanopus is not only obligate, but also demographically rigid and easily perturbed by anemonefish fisheries. Clearly, these two species must be managed together as a unit and with utmost precaution. To this end, we propose several tangible management actions that will help to minimize fishing effects.     

A jellyfish-eating sea anemone (Cnidaria,Actiniaria) from Palau: Entacmaea medusivora sp. nov.

Entacmaea medusivora sp. nov., a member of the family Actiniidae, inhabits Jellyfish Lake in Palau, western Caroline Islands, and eats the rhizostome jellyfish Mastigias papua. The anemone is azooxanthellate, despite its jellyfish prey containing symbiotic algae. Well fed anemones released ciliated planula-like larvae in the laboratory when maintained at more than 30 °C. It could not be determined whether the larvae, which usually settled around the parent within 24 h of release, were produced sexually or asexually.     

Bioactive Compounds of Sea Anemones: A Review

Marine organisms and their associated microorganisms contain a wide range of novel bioactive natural compounds that are widely used in the field of anti-microbial, anti-tumor, and anti-cancer drug discovery research. Hence, much focus has been given to isolate the bioactive compounds from marine sources. Sea anemone, one such marine resource, is used in recent years to extract bioactive compounds. It belongs to the phylum Cnidaria. The distinguishing feature of cnidarians is nematocysts, specialized venomous organs that the animals use mainly for capturing prey and protecting themselves from predators. There are over one thousand species of sea anemone reported worldwide and of which 40 species belonging to 17 families are found in India. Out of 40 species, 24 are marine, 13 are estuarine and 3 are common to both habitats. We present an overview of some of the potential marine bioactive compounds from a curative point of view isolated from sea anemone. Among the Order Actiniaria, Family Actiniidae exhibits by far the highest number of species yielding promising compounds, followed by Family Stichodactylidae. Haemolytic activity has been the major area of interest in the screening of actinarian compounds.

     

Patterns of pair formation in protandrous anemonefishes,Amphiprion clarkii,A. frenatus andA. perideraion,on coral reefs of Okinawa,Japan

Synopsis At Sesoko Island, stocks of anemonefishes were observed for 2 years in a 350 m × 150 m area where host sea anemones were sparse. About 40% of the pairs separated, mostly due to typhoon attacks or displacement. Widowed mates remained and acquired new mates on the same sea anemone, except for one case. New mates were immigrant adults inAmphiprion clarkii and A.frenatus, but resident juveniles inA. perideraion. A. clarkii andA. frenatus moved between sea anemones and sometimes displaced smaller consexuals, butA. perideraion rarely moved. The difference in mobility among the 3 species was related to the mean difference in standard length between the largest juveniles in breeding groups and the minimum size of breeding males in each species, and that between breeding males and the minimum size of breeding females. InA. perideraion both of these differences were small, so pairs could be formed quickly by residents after mate loss. On the contrary, inA. clarkii andA. frenatus, either or both differences were large and it would take a longer time to form a pair after mate loss. Differences in mobility affected the patterns of pair formation, and then the size composition of members in breeding groups.     

Effects of anemonefish on giant sea anemones: expansion behavior,growth, and survival

The symbiosis between giant sea anemones and anemonefish on coral reefs is well known, but little information exists on impacts of this interaction on the sea anemone host. On a coral reef at Eilat, northern Red Sea, individuals of the sea anemone Entacmaea quadricolor that possessed endemic anemonefish Amphiprion bicinctus expanded their tentacles significantly more frequently than did those lacking anemonefish. When anemonefish were experimentally removed, sea anemone hosts contracted partially. Within 1–4 h in most cases, individuals of the butterflyfish Chaetodon fasciatus arrived and attacked the sea anemones, causing them to contract completely into reef holes. Upon the experimental return of anemonefish, the anemone hosts re-expanded. The long-term growth rate and survival of the sea anemones depended on the size and number of their anemonefish. Over several years, sea anemones possessing small or no fish exhibited negative growth (shrinkage) and eventually disappeared, while those with at least one large fish survived and grew. We conclude that host sea anemones sense the presence of symbiotic anemonefish via chemical and/or mechanical cues, and react by altering their expansion behavior. Host sea anemones that lack anemonefish large enough to defend them against predation may remain contracted in reef holes, unable to feed or expose their tentacles for photosynthesis, resulting in their shrinkage and eventual death.     

Host Imprinting in Anemonefishes (Pisces: Pomacentridae): Does it Dictate Spawning site Preferences?

The olfactory cues, to which some species of anemonefish embryos imprint, are secreted in the mucus on the tentacles and the oral disc of the host anemone. Close contact of the eggs of anemonefishes with the host's tentacles seems therefore important to imprinting. A corollary of this observation is that if local environmental conditions sweep tentacles in one specific direction, then the eggs will be placed leeward of the tentacles, rather than to foremost way from the tentacles. Other known factors such as egg predation can also cause a spawning site preference. No study has examined the possibility of the existence of such a preference. In this study, we addressed two questions: (1) Does spawning site preference exist in anemonefishes? (2) If yes, is it possible to relate this to the imprinting hypothesis, i.e. does local ocean currents over the host anemone have any influence on this preference? Two different coral reef areas were surveyed for anemonefish groups with eggs present: Eilat and Na'ama in the Aquaba-bight, the Red Sea (RS), and areas at Lizard Island, the northern Great Barrier Reef (GBR). We found the anemonefishes Amphiprion akindynos (GBR), A. bicinctus (RS), A. melanopus (GBR), and A. perideraion (GBR), to have a distinctive spawning site preference. We discuss the relevance of these findings to anemonefish host imprinting.     

Flexibility of nutritional strategies within a mutualism: food availability affects algal symbiont productivity in two congeneric sea anemone species

Mutualistic symbioses are common, especially in nutrient-poor environments where an association between hosts and symbionts can allow the symbiotic partners to persist and collectively out-compete non-symbiotic species. Usually these mutualisms are built on an intimate transfer of energy and nutrients (e.g. carbon and nitrogen) between host and symbiont. However, resource availability is not consistent, and the benefit of the symbiotic association can depend on the availability of resources to mutualists. We manipulated the diets of two temperate sea anemone species in the genus Anthopleura in the field and recorded the responses of sea anemones and algal symbionts in the family Symbiodiniaceae to our treatments. Algal symbiont density, symbiont volume and photosynthetic efficiency of symbionts responded to changes in sea anemone diet, but the responses depended on the species of sea anemone. We suggest that temperate sea anemones and their symbionts can respond to changes in anemone diet, modifying the balance between heterotrophy and autotrophy in the symbiosis. Our data support the hypothesis that symbionts are upregulated or downregulated based on food availability, allowing for a flexible nutritional strategy based on external resources.     

Molecular phylogenetic evidence for the evolution of specialization in anemonefishes.

Anemonefishes (genera: Amphiprion and Premnas; family Pomacentridae) are a group of 28 species of coral reef fishes that are found in obligate symbiosis with large tropical sea anemones. A phylogenetic hypothesis based on morphological analyses of this group suggests that the ancestral anemonefish was a generalist with similar morphology to other pomacentrids, and that it gave rise to other anemonefish species that were more specialized for living with particular species of host anemones. To test this hypothesis we constructed a molecular phylogeny for the anemonefishes by sequencing 1140 base pairs of the cytochrome b gene and 522 base pairs of the 16S rRNA gene for six species of anemonefishes (representatives of all subgenera and species complexes) and two other pomacentrid species. Three methods of phylogenetic analysis all strongly supported the conclusion that anemonefishes are a monophyletic group. The molecular phylogeny differs from the tree based on morphological data in that the two species of specialized anemonefishes (Premnas biaculeatus and Amphiprion ocellaris) were assigned to a basal position within the clade, and the extreme host generalist (Amphiprion clarkii) to a more derived position. Thus, the initial anemonefish ancestors were probably host specialists and subsequent speciation events led to a combination of generalist and specialist groups. Further phylogenetic studies of additional anemonefish species are required to substantiate this hypothesis.     

Marine protected area restricts demographic connectivity: Dissimilarity in a marine environment can function as a biological barrier

The establishment of marine protected areas (MPAs) can often lead to environmental differences between MPAs and fishing zones. To determine the effects on marine dispersal of environmental dissimilarity between an MPA and fishing zone, we examined the abundance and recruitment patterns of two anemonefishes (Amphiprion frenatus and A. perideraion) that inhabit sea anemones in different management zones (i.e., an MPA and two fishing zones) by performing a field survey and a genetic parentage analysis. We found lower levels of abundance per anemone in the MPA compared to the fishing zones for both species (n = 1,525 anemones, p = .032). The parentage analysis also showed that lower numbers of fishes were recruited from the fishing zones and outside of the study area into each anemone in the MPA than into each anemone in the fishing zones (n = 1,525 anemones, p < .017). However, the number of self‐recruit production per female did not differ between the MPA and fishing zones (n = 384 females, p = .516). Because the ocean currents around the study site were unlikely to cause a lower settlement intensity of larvae in the MPA, the ocean circulation was not considered crucial to the observed abundance and recruitment patterns. Instead, stronger top‐down control and/or a lower density of host anemones in the MPA were potential factors for such patterns. Our results highlight the importance of dissimilarity in a marine environment as a factor that affects connectivity.     

Why do anemonefishes inhabit only some host actinians?

Synopsis The 25 species ofAmphiprion and one ofPremnas (family Pomacentridae) are obligate symbionts of 10 species of facultatively symbiotic sea anemones. Throughout the tropical Indo-West Pacific range of the relationship, a fish species inhabits only certain of the hosts potentially available to it. This specificity is due to the fishes. Five fishes occupy six sea anemone species at Lizard Island, Great Barrier Reef, Australia.Entacmaea quadricolor harborsP. biaculeatus, A. melanopus andA. akindynos. Adults ofPremnas occur deeper than about 3 m in large, primarily solitary actinians; juveniles may occupy peripheral members ofEntacmaea clones in shallow water. Specimens ofA. melanopus live exclusively in clonal anemones, which are found no deeper than 3 m. Most individuals ofA. akindynos inEntacmaea are juveniles, occurring shallow and deep, in solitary anemones or at the margins of clones. Interspecific as well as intraspecific social control of growth may be responsible for keeping fish small at clone fringes. Conspicuous specimens ofE. quadricolor depend upon their anemonefish to survive. Actinians cleared of symbionts disappeared within 24 h, probably having been eaten by reef fishes.Entacmaea, the most abundant and widespread host actinian at Lizard Island and throughout the range of the association, is also arguably the most attractive to anemonefishes. I believe its vulnerability to predation was a factor in its evolving whatever makes it desirable to fishes. Experimental transfers pitted fish of one species against those of another, controlling for ecophenotype of host, and sex, size and number of fish. Competitive superiority was in the same order as abundance and over-all host specificity:P. biaculeatus, A. melanopus, A. akindynos. At least three factors are necessary to explain patterns of species specificity - innate or learned host preference, competition, and stochastic processes.     

Protection of host anemones by snapping shrimps: a case for symbiotic mutualism?

The sea anemone Bartholomea annulata is an ecologically important member of Caribbean coral reefs which host a variety of symbiotic crustacean associates. Crustacean exosymbionts typically gain protection from predation by dwelling with anemones. Concurrently, some symbionts may provide protection to their host by defending against anemone predators such as the predatory fireworm, Hermodice carunculata, which can severely damage or completely devour prey anemones. Herein we show through both field and laboratory studies that anemones hosting the symbiotic alpheid shrimp Alpheus armatus are significantly less likely to sustain damage by H. carunculata than anemones without this shrimp. Our results suggest that the association between A. armatus and B. annulata, although complex because of the numerous symbionts involved, may be closer to mutualism on the symbiotic continuum.     

Isolation, characterization, and amino acid sequence of a polypeptide neurotoxin occurring in the sea anemone Stichodactyla helianthus

An aqueous exudate collected from frozen and thawed bodies of a Caribbean sea anemone, Stichodactyla (formerly Stoichactis) helianthus, contained a polypeptide neurotoxin (Sh I) selectively toxic to crustaceans. The polypeptide was purified by G-50 Sephadex, phosphocellulose, and sulfopropyl-Sephadex chromatography and shown to have a molecular size of 5200 daltons and a pI of 8.3. The amino acid sequence determined by automatic Edman degradations of whole RCM Sh I and of its clostripain, staphylococcal protease, and cyanogen bromide digest peptides is A1ACKC5DDEGP10DIRTA15PLTGT20VDLGS25CNAGW30EKCAS35YYTII40ADCCR45KKK . Only 33% of this sequence is identical with the sequence of Anemonia sulcata toxin II, a sea anemone toxin isolated from the taxonomic family Actiniidae. The six half-cystines are located in equivalent positions to those of the actiniid toxins and account for nearly half of the residues common to all of the toxins. However, 69% of the Sh I sequence is identical with that of toxin II from Heteractis paumotensis, another sea anemone belonging to the family Stichodactylidae. Stichodactylid toxins lack the initial N-terminal residue of actiniid toxins and possess three consecutive acidic residues at positions 6-8, a single tryptophan at position 30, and four consecutive basic residues at positions 45-48 (C-terminus). A rabbit IgG prepared by Sh I immunization bound Sh I with a K0.5 of 4.7 nM but failed to bind homologous actiniid (Anemonia sulcata II, Condylactis gigantea III) or bolocerid (Bolocera tuedae II) polypeptide neurotoxins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kunitz-type protease inhibitors from acrorhagi of three species of sea anemones

Sea anemones are rich in biologically active polypeptides such as toxins and protease inhibitors. These polypeptides have so far been isolated from whole bodies, tentacles or secreted mucus. Recently, two novel peptide toxins with crab lethality have been isolated from acrorhagi (specialized aggressive organs elaborated by only certain species of sea anemones belonging to the family Actiniidae) of Actinia equina. This prompted us to survey biologically active polypeptides in the acrorhagi of two species of sea anemones, Anthopleura aff. xanthogrammica and Anthopleura fuscoviridis. No potent crab lethality was displayed by the acrorhagial extracts of both species. However, significantly high protease inhibitory activity was instead detected in the acrorhagial extracts of the two species and also in that of A. equina. From the acrorhagi of A. equina, A. aff. xanthogrammica and A. fuscoviridis, one (AEAPI), one (AXAPI) and two (AFAPI-I and AFAPI-III) protease inhibitors were isolated, respectively. The complete amino acid sequences of the four inhibitors were elucidated by N-terminal sequencing and sequencing of the C-terminal peptide fragment produced upon asparaginylendopeptidase digestion. The determined amino acid sequences revealed that all the four inhibitors are new members of the Kunitz-type protease inhibitor family.