The chemical defenses of a stylocellid (Arachnida, Opiliones, Stylocellidae) from Sulawesi with comparisons to other Cyphophthalmi.
Abstract: Two female specimens of an undescribed species of stylocellid harvestman (Opiliones, Stylocellidae) from Sulawesi were extracted in methanol, and compounds in the product were identified by means of gas chromatography-mass spectrometry. Nineteen significant peaks were found, 12 of which were identified, indicating the presence of naphthoquinone, 2-tridecanone, 2-tetradecanone, 2-pentadecanone, 6-methyl-1,4-naphthoquinone, 6-methyl-1,4- naphthalenediol, and at least four unsaturated ketones. The spectrum differed both qualitatively and quantitatively from previously published data on Siro exilis Hoffman 1963 and Cyphophthalmus duricorius Joseph 1868 (Sironidae).

Keywords: Siro, Cyphophthalmus, exocrine products, ketones, naphthoquinones
Article Type: Report
Subject: Harvestmen (Physiological aspects)
Harvestmen (Behavior)
Harvestmen (Comparative analysis)
Animal defenses (Chemical properties)
Animal defenses (Physiological aspects)
Animal defenses (Comparative analysis)
Authors: Jones, Tappey H.
Shear, William A.
Giribet, Gonzalo
Pub Date: 05/01/2009
Publication: Name: Journal of Arachnology Publisher: American Arachnological Society Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2009 American Arachnological Society ISSN: 0161-8202
Issue: Date: May, 2009 Source Volume: 37 Source Issue: 2
Topic: Canadian Subject Form: Animal defences; Animal defences; Animal defences
Geographic: Geographic Scope: Indonesia Geographic Code: 9INDO Indonesia
Accession Number: 238834220
Full Text: Harvestmen (Opiliones) have a distinctive pair of prosomal exocrine glands opening to the surface via ozopores usually placed in the vicinity of the second leg pair. These glands produce a variety of substances (Table 1) effective in defense (e.g., Juberthie 1961a, 1961b, 1976; Eisner et al. 1971; Jones et al. 1977), and possibly with other functions as well. The secretions of approximately 48 species have been studied in detail up to this time. The results have been effectively reviewed by Gnaspini & Hara (2007; also see Hara et al. 2005), who presented a table listing the species and the compounds recorded from them. Forty-six compounds have been identified to date (numerous others are present but have not been identified), falling into the broad classes of long-chain alcohols and ketones, hydroxyquinones, phenols, and, more rarely, an alkaloid (nicotine), an amine (N,N-dimethyl-B-phenylethylamine), terpenoids (camphene, limonene) and bornyl esters (bornyl acetate and propionate) (Gnaspini & Hara 2007). A number of these compounds are unique or rare in nature, particularly in animals. Typically a single species produces a mixture of compounds, and the mixture of both identified and unidentified molecules may be characteristic of the particular taxon (family, genus, species) involved (Hara et al. 2005).

Although the defensive chemistry of Cyphophthalmi, Eupnoi and Laniatores has been studied for some species, that of many important higher taxa (i.e., Dyspnoi) remains completely uninvestigated. Our preliminary results (unpublished data) suggest that travunioids, only a single species of which has been examined so far, are extraordinarily diverse in their chemistry. Studies of Grassatores have concentrated on just a few families (Cosmetidae, Gonyleptidae, Manaosbiidae, Stygnommatidae) in this very diverse assemblage.

Defensive chemistry of the basal and divergent harvestman suborder Cyphophthalmi (e.g., see Giribet et al. 2002 for the phylogenetic placement of Cyphophthalmi) is interesting because if the composition of defensive secretions is of any phylogenetic use, its ancestral state in this group could be used to polarize the characters higher in the tree and to optimize the ancestral state of the defensive substances in Opiliones. But to date, only two cyphophthalmid species, Siro exilis Hoffman 1963 of North America, and Cyphophthalmus duricorius Joseph 1868 of Europe, have been studied for their defensive secretions (Raspotnig et al. 2005). Both of these species belong to the same family, Sironidae, while members of the other extant five families remain unexamined. The secretions of the two sironids consist of complex arrays of ketones and naphthoquinones.

In this paper, we present data on the secretion of an undescribed species probably belonging to the genus Stylocellus, family Stylocellidae. This species will be named and described in a forthcoming revision of the family by Ronald Clouse. In the current phylogenies of cyphophthalmids, the families Stylocellidae or Pettalidae appear basal within the suborder (Giribet & Boyer 2002; Boyer et al. 2007), although their stable placement is still debatable. The family Stylocellidae is found exclusively in Southeast Asia, from Southern China and Northeast India to the western side of New Guinea, in the Indonesian province of Irian Jaya, and the Philippine island of Palawan (Shear 1993; Clouse & Giribet 2007; Giribet et al. 2007; R. Clouse, in progress). The species we studied was collected on the Indonesian island of Sulawesi and is tentatively assigned to the genus Stylocellus, although the whole generic systematics of the family is currently under reexamination (R. Clouse & G. Giribet, in progress).

METHODS

Two female Stylocellus specimens of a yet undescribed species (Fig. 1) were collected alive in Bantimurung, Sulawesi (5[degrees]02'33"S, 119[degrees]44'08"E; 348 m elev.; Giribet locality number 512; collected 28 June 2008, R. Clouse, G. Giribet, C. Rahmadi, leg.; MCZ DNA101938), shipped alive to WAS, and extracted in about 0.5 ml of USP methanol. The extracts of the two specimens were pooled. The code number 06-172 was assigned to the specimens, both of which will be placed as vouchers in the Museum of Comparative Zoology, Cambridge, Massachusetts. The analysis of the extract was performed by THJ. Gas chromatography-mass spectrometry was carried out in the EI mode using a Shimadzu QP-5000 GC/MS equipped with an RTX-5, 30 m X 0.25-mm i.d. column. The instrument was programmed from 60[degrees] C to 250[degrees] Cat10[degrees]/min. Identification of components was accomplished using NIST/EPA/NIH mass spectral library on CD-rom, version 1.7 (1999) and the NIST/EPA/NIH mass spectral library version 2.0d (2005).

[FIGURE 1 OMITTED]

RESULTS AND DISCUSSION

After gas chromatography-mass spectrometry examination, 19 significant peaks were identified in the extract. The major peaks in our analysis indicate the presence of naphthoquinone, 2-tridecanone, 2-tetradecanone, 2-pentadecanone, 6-methyl1,4-naphthoquinone, 6-methyl-1,4-naphthalenediol, and at least four unsaturated ketones, two of which correspond to peaks S and U as noted by Raspotnig et al. (2005), so we have adopted their identification of these compounds (Table 2).

Table 3 compares the analyses of the three species of Cyphophthalmi. Chloronaphthoquinones are unique in the two sironid species (and as exocrine products of arthropods [Raspotnig et al. 2005]), but were not found in the stylocellid species; likewise undecan-2-one, 6-tridecen-2-one and 7-tridecen-2-one were absent from the secretion of Stylocellus. It is possible that these unusual compounds came from the males included in the sironid samples. Furthermore, the stylocellid secretion contained 6-methyl-1,4-naphthalenediol, a reduction product of 6-methyl-1,4-naphthoquinoine, which did not occur in the two sironids. The percent composition of compounds that were common to the three species shows strong differences between the stylocellid and the two sironids, and to a lesser degree, between Siro exilis and Cyphophthalmus duricorius. While 2-tridecanone made up about 20% of the secretion of both sironids, it comprised 50% in the stylocellid; 1,4-naphthoquinone and 6-methyl-1,4-naphthoquinone were found in very small amounts in the stylocellid, but at values from about 12% to nearly 18% in the sironids; pentadecan-2-one was at 13.8% for the stylocellid, butless than1.7% for the sironids, and so on.

In summary, we may observe that while the composition of the secretions of the three species is similar in constituting mixtures of ketones and naphthoquinones, there are significant differences both qualitative and quantitative. Some of the differences could be due to the fact that we extracted our animals in methanol, while Raspotnig et al. (2005) used hexane. Raspotnig et al. (2005) found that the composition of the secretions in the two species studied by them was highly consistent from individual to individual within species, as has been reported in numerous previous studies of harvestman defensive secretions. This lessens our concern about the small size of our sample versus the much larger samples taken by Raspotnig et al. (2005); the remote location and relative rarity of the animals we studied makes it unlikely that large samples will be available in the near future.

The secretion of the stylocellid seems to contain fewer compounds than found by Raspotnig et al. (2005), who identified at least tentatively all of the 24 peaks they found (as opposed to our 19). However, very small peaks were not considered by us. Nevertheless, the observation about the number of compounds would hold on the basis of the number of peaks alone. Aside from 5-methylnaphthoquinone, not found in the sironids, the stylocellid secretion is strongly dominated by ketones and lacks the diversity of naphthoquinones found in sironids. If stylocellids are in fact a sister group to other Cyphophthalmi, this observation suggests that the other naphthoquinones, especially the chlorinated ones, have been added in the course of evolution (or lost in the stylocellid lineage), and perhaps that ketones dominated the secretion of the common ancestor of extant Opiliones. Quinones may have been co-opted later from substances used by all arthropods to sclerotize cuticular proteins. However, proper polarization of this character requires first, a well-resolved cyphophthalmid phylogeny and second, a proper outgroup comparison. Since Opiliones' putative outgroups (Solifugae, Scorpiones, and Pseudoscorpiones; e.g., Wheeler & Hayashi 1998; Giribet et al. 2002; Shultz 2007) do not have this type of secretion, polarization may be difficult.

[FIGURE 2 OMITTED]

Raspotnig et al. (2005) pointed out that while both ketones and naphthoquinones are found in Cyphophthalmi, sclerosomatids (suborder Eupnoi) secrete only ketones (and alcohols) and the single phalangiid studied (also an Eupnoi), naphthoquinones. In Laniatores, Grassatores utilize phenols and methylquinones, while the single member of Insidiatores analyzed uses an eclectic mix of N.N-dimethyl-B-phenylethylamine, nicotine, bornyl esters, and terpenes. But again we must point out the strong bias in the data. Most of the species studied have been either Grassatores or sclerosomatids (see Table 1), while Dyspnoi and the Eupnoi family Caddidae, two groups of significant phylogenetic importance, have not been studied chemically at all.

In addition, no study has yet been made of the effect of collection method on the results of the analysis of harvestman secretions. In our studies, we are using methanol to extract whole bodies of live specimens, but other solvents, such as hexane, have been used in other laboratories. In some studies, live animals are induced to secrete their defensive compounds, which are collected either by micropipettes, fine glass tubing, or by absorption on filter paper. Thus we do not know if differences between species where different collection methods were used are real, or artifacts of the different methods. Certainly the fact that in many species the secretion of the repugnatorial glands is mixed with regurgitate from the gut could play a role if the already-mixed secretion is collected. The question of sample size also arises; here we used pooled extract from only two animals of the same sex, while Raspotnig et al (2005) used large samples including both sexes and juveniles, and analyzed each extract individually. Previous studies of harvestman defensive secretions vary greatly as to the numbers of individuals sampled, and in some cases numerous animals were used, but the samples were pooled for analysis. In the near future we hope to carry out a study using different collection methods and solvents on numerous individuals of the same species, in order to compare the effects of these methods.

Hara et al. (2005) were able to map the composition of the secretions of 22 gonyleptids (Laniatores, Grassatores) on a phylogenetic tree constructed from other characters. They found rampant homoplasy, but noted that closely related compounds (pairs of phenols and quinones) could easily be transformed into one another by oxidation or reduction, suggesting that "families" of such compounds could represent synapomorphies as transformation series of compound families. However, their final conclusions were ambiguous. On the one hand, it appeared that the great diversity of the secretions did not allow the recognition of phylogenetic lineages, with some exceptions, but on the other, they hoped that the analysis of more species and the addition of metabolic sequences and interchangeable compounds might provide phylogenetic information in the future. We agree with this position and plan to continue to analyze opilionid defensive secretions not only in a search for molecules new to science or new to arthropods, but also with the expectation that more data will help to clarify phylogenetic relationships within Opiliones.

ACKNOWLEDGMENTS

Ron Clouse and Cahyo Rahmadi participated in the collecting trip to Sulawesi that yielded the specimens for this study. Support for fieldwork was made possible by a Putnam Grant from the Museum of Comparative Zoology. Permits were awarded by LIPI. The work of WAS was supported by a grant from the Professional Development Committee of Hampden-Sydney College.

Manuscript received 11 June 2008, revised 12 January 2009.

LITERATURE CITED

Boyer, S.L., R.M. Clouse, L.R. Benavides, P.J. Schwendinger, I. Kuranarathna & G. Giribet. 2007. Biogeography of the world: a case study from cyphophthalmid Opiliones, a globally distributed group of arachnids. Journal of Biogeography 34:2070-2085.

Clouse, R.M. & G. Giribet. 2007. Across Lydekker's Line--first report of mite harvestmen (Opiliones : Cyphophthalmi: Stylocellidae) from New Guinea. Invertebrate Systematics 21:207-227.

Eisner, T., A.F. Kluge, J.E. Carrel & J. Meinwald. 1971. Defense of phalangid: liquid repellent administered by leg dabbing. Science 173:650-652.

Giribet, G. & S.L. Boyer. 2002. A cladistic analysis of the cyphophthalmid genera (Opiliones, Cyphophthalmi). Journal of Arachnology 30:110-128.

Giribet, G., G.D. Edgecombe, W.C. Wheeler & C. Babbitt. 2002. Phylogeny and systematic position of Opiliones: a combined analysis of chelicerate relationships using morphological and molecular data. Cladistics 18:5-70.

Giribet, G., P.P. Sharma & D.B. Bastawade. 2007. A new genus and species of Cyphophthalmi (Arachnida: Opiliones) from the northeastern states of India. Zoological Journal of the Linnean Society 151:663-670.

Gnaspini, P. & M.R. Hara. 2007. Defense mechanisms. Pp. 374-399. In Harvestmen: The Biology of Opiliones. (R. Pinto-Da-Rocha, G. Machado & G. Giribet, eds.). Harvard University Press, Cambridge, MA.

Hara, M.R., A.J. Cavalheiro, P. Gnaspini & D.Y.A.C. Santos. 2005. A comparative analysis of the chemical nature of defensive secretions of Gonyleptidae (Arachnida: Opiliones: Laniatores). Biochemical Systematics and Ecology 33:1210-1225.

Jones, T.H., J. Meinwald, K. Hicks & T. Eisner. 1977. Characterization and synthesis of volatile compounds from the defensive secretions of some "daddy longlegs" (Arachnida: Opiliones: Leiobunum spp.). Proceedings of the National Academy of Sciences USA 74:419-422.

Juberthie, C. 1961a. Structure des glandes odorantes et modalites d'utilisation de leur secretion chez deux Opilions Cyphophthalmes. Bulletin de la Societe zoologique de France 86:106-116.

Juberthie, C. 1961b. Structure et fonction des glandes odorantes chez quelques Opilions (Arachnida). Verhandlungen der Deutschen Zoologischen Gesellschaft in Saarbriicken 1961:533-537.

Juberthie, C. 1976. Chemical defence in soil Opiliones. Revue d'Ecologie et de Biologie du Sol 13:155-160.

Raspotnig, G., G. Fauler, M. Leis & H.J. Leis. 2005. Chemical profiles of scent gland secretions in the cyphophthalmid opilionid harvestmen, Siro duricorius and S. exilis. Journal of Chemical Ecology 31:1353-1368.

Shear, W.A. 1993. New species in the opilionid genus Stylocellus from Malaysia, Indonesia and the Philippines (Opiliones, Cy phophthalmi, Stylocellidae). Bulletin of the British Arachnological Society 9:174-188.

Shultz, J.W. 2007. A phylogenetic analysis of the arachnid orders based on morphological characters. Zoological Journal of the Linnean Society 150:221-265.

Wheeler, W.C. & C.Y. Hayashi. 1998. The phylogeny of extant chelicerate orders. Cladistics 14:173-192.

Tappey H. Jones (1), William A. Shear (2,4) and Gonzalo Giribet (3)

(1) Department of Chemistry, Virginia Military Institute, Lexington, VA 24450, USA; (2) Department of Biology, Hampden-Sydney College, Hampden-Sydney, VA 23901, USA; (3) Museum of Comparative Zoology, Harvard University, Cambridge, MA 02138, USA (4) Corresponding author. E-mail: wshear@hsc.edu
Table 1.-Distribution of defensive secretions in Opiliones. For
more detail, see Gnaspini & Hara (2007).

Taxon                       Compounds

Suborder Cyphophthalmi      Ketones, naphthoquinones
Suborder Eupnoi
  Family Phalangiidae       naphthoquinones
  Family Sclerosomatidae    ketones, alcohols
Suborder Dyspnoi            unknown
Suborder Laniatores
  Infraorder Insidiatores   Amines, bornyl esters, terpenes,
                              alkaloids
                            Phenols, methylquinones
  Infraorder Grassatores    Benzoquinones, phenols

Table 2.-Compounds and percent composition identified in methanol
extract of whole females of the stylocellid from Sulawesi. See also
Fig. 2.

Peak   Compound                        Relative %      Peak code in
                                                     Raspotnig, et al.
                                                           2005

1      1,4-Naphthoquinone                   2                E
2      2-Tridecanone                       42                J
3      6-methylnaphthoquinone               1                L
4      Methyl branched 2-tridecanone        8             M or N
5      2-Tetradecanone                      9             M or N
6      6-Methyl-1,4-naphthalenediol         1          Not detected
7      2-Pentadecadienone                   8                S
8      2-Pentadecenone                      7                U
9      Unsaturated ketone a                 3                ?
10     Unsaturated ketone b                 3                ?
11     2-Pentadecanone                     14                W
12     2-Methoxy-1,4-naphthoquinone         3          Not detected

Table 3.-Comparison of percent composition of secretions of the
stylocellid from Sulawesi with those of Siro exilis and Cyphophthalmus
duricorius (data from Raspotnig et al. 2005). Confidence limits are
given for the data on S. exilis and C. duricorius because Raspotnig et
al. (2005) were reporting on individual extractions from 26 and 95
adult specimens respectively (for a complete list of compounds
identified from the sironid species, see Raspotnig et al. [2005]). Our
measurements are single points representing a pooled extraction of two
adults due to the smaller collections of stylocellids when compared to
the two sironid species studied previously. Bold figures represent the
largest amount if significant differences are present; if two figures
in a row are in bold type, there was no statistically significant
difference between those two.

              Compound                  Stylocellid

2-Tridecanone                               50.0
7-Tridecen-2-one                       not identified
1,4-Naphthoquinone                          1.7
6-Methyl-1,4-naphthoquinone                 1.0
Undecan-2-one                          not identified
4-Chloro-1,2-naphthoquinone            not identified
6-Tridecen-2-one                       not identified
Pentadecan-2-one                            13.8
6-Methyl-4-chloro-1,2-naphthoquinone   not identified
2-Tetradecanone                             8.0
Pentadecadieone                             8.0
Pentadecenone                               6.7
6-Methyl-1,4-naphthalenediol                0.7

              Compound                      S. exilis

2-Tridecanone                          20.28 [+ or -] 3.79
7-Tridecen-2-one                       15.47 [+ or -] 1.35
1,4-Naphthoquinone                     14.01 [+ or -] 1.9
6-Methyl-1,4-naphthoquinone            13.08 [+ or -] 1.43
Undecan-2-one                           0.57 [+ or -] 0.19
4-Chloro-1,2-naphthoquinone            11.83 [+ or -] 1.68
6-Tridecen-2-one                        4.13 [+ or -] 0.92
Pentadecan-2-one                        1.68 [+ or -] 0.5
6-Methyl-4-chloro-1,2-naphthoquinone    4.30 [+ or -] 1.24
2-Tetradecanone                         1.10 [+ or -] 0.33
Pentadecadieone                         3.0 [+ or -] 0.86
Pentadecenone                           4.5 [+ or -] 1.14
6-Methyl-1,4-naphthalenediol              not identified

              Compound                    C. duricorius

2-Tridecanone                          20.21 [+ or -] 3.56
7-Tridecen-2-one                       18.98 [+ or -] 2.38
1,4-Naphthoquinone                     17.61 [+ or -] 2.82
6-Methyl-1,4-naphthoquinone            12.15 [+ or -] 2.03
Undecan-2-one                           9.71 [+ or -] 1.59
4-Chloro-1,2-naphthoquinone             7.09 [+ or -] 2.44
6-Tridecen-2-one                        4.02 [+ or -] 1.03
Pentadecan-2-one                        0.01 [+ or -] 0.02
6-Methyl-4-chloro-1,2-naphthoquinone    0.36 [+ or -] 0.51
2-Tetradecanone                         0.65 [+ or -] 0.25
Pentadecadieone                         0.04 [+ or -] 0.06
Pentadecenone                           0.03 [+ or -] 0.05
6-Methyl-1,4-naphthalenediol              not identified
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