Life cycle of Sarraceniopus nipponensis (Histiostomatidae: Astigmata) from the fluid-filled pitchers of alata (Sarraceniaceae) J. Krüger, S. Wirth

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J. Krüger, S. Wirth. Life cycle of Sarraceniopus nipponensis (Histiostomatidae: Astigmata) from the fluid-filled pitchers of Sarracenia alata (Sarraceniaceae). Acarologia, Acarologia, 2011, 51(2), pp.259-267. ￿10.1051/acarologia/20112008￿. ￿hal-01600022￿

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Acarologia is under free license and distributed under the terms of the Creative Commons-BY-NC-ND which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Acarologia 51(2): 259–267 (2011) DOI: 10.1051/acarologia/20112008

LIFE CYCLE OF SARRACENIOPUS NIPPONENSIS (HISTIOSTOMATIDAE: ASTIGMATA) FROM THE FLUID-FILLED PITCHERS OF SARRACENIA ALATA (SARRACENIACEAE)

Jessica KRÜGER and Stefan WIRTH*

(Received 10 December 2010; accepted 18 April 2011; published online 30 June 2011)

Museum für Naturkunde, Leibniz-Institut für Evolutions- und Biodiversitätsforschung, an der Humboldt-Universität zu Berlin, Invalidenstraße 43, 10115 Berlin, Germany. [email protected], [email protected] ( *corresponding author)

ABSTRACT — Specimens of a population of Sarraceniopus nipponensis collected from Sarracenia alata in Louisiana, USA, slightly differ morphologically from specimens of the type location in Japan. Hence, a subspecies level for the USA- population is proposed. Ecological and behavioral data of S. nipponensis (Louisiana, USA) were collected. The develop- ment from larva to adult, skipping the facultative deutonymphal stage, takes about 6 days at an average temperature of 24°C (room temperature). Adults show a sex ratio of 1 : 2.6 in favor of females. Deutonymphs have a partially re- duced sucker plate, apparently dispersing through ambulatory activity rather than phoresy. Precopulatory mate guard- ing occurs many hours before tritonymphs enter quiescence. During mate guarding, males rest motionlessly on the tritonymphal dorsum, but are not unable to move meanwhile. Legs I are conspicuously thick and serve as ’safeguard holder’ while legs II are used to fight against male competitors and to hold on to the female regardless of environmental disturbances. The precopulated tritonymph is held in position directly beneath the male. KEYWORDS — Sarraceniopus nipponensis; Histiostomatidae; life cycle; Sarracenia alata; ecology; subspecies; Kisatchie Na- tional Forest; USA-Louisiana

INTRODUCTION duce a digestive fluid, which forms a diverse mi- crohabitat for different kinds of such as Different kinds of habitats are colonized by the the histiostomatid species: Sarraceniopus nip- histiostomatids (Hunter and Hunter 1964; Nesbitt ponensis Tagami, 2004 (Tagami 2004). Fashing and 1954), such as the fluid-filled pitchers of plants be- OConnor (1984) and Wirth (2004) proposed that longing to and Sarracenia. Mite species the group of histiostomatid "pitcher-plant-" is of Creutzeria, Nepenthacarus and Zwickia live in the monophyletic. pitchers of old world Nepenthes, while mite species of the genus Sarraceniopus are present on the New Although mites are commonly found in such World Sarracenia (endemic to the South and South- watery habitats, the biology of most of them is East of the USA) which convergently developed poorly known (Fashing 2004a). The phenomenon similar pitchers as those observed in species of of mate guarding (pre-copulation) is common in the genus Nepenthes. These pitcher plants pro- histiostomatid mites. However, within the mono- http://www1.montpellier.inra.fr/CBGP/acarologia/ 259 ISSN 0044-586-X (print). ISSN 2107-7207 (electronic) Krüger J. and Wirth S.

FIGURE 1: Schematized life cycle of Sarraceniopus nipponensis (USA) – big cycle: obligate cycle, small cycle: optional part with molting into deutonymphs, grey: ecdysis stages, slightly grey: active developmental stage. The first ecdysis from prelarva to larva occurs within the egg. Development from larva to adult without forming a deutonymph takes on average 6 days for both sexes. Sex ratio males : females ≈ 1 : 2.6.

260 Acarologia 51(2): 259–267 (2011) phyletic pitcher plant group, it has only been stud- ration of mites. A total of 85 single larvae were iso- ied for Zwickia sp. and Sarraceniopus darlingtoniae lated, 38 of them developed until adulthood. Be- Fashing and OConnor, 1984 (Fashing 2004a,b). This cause these specimens needed a complex micro- behavioral pattern is assumed to ensure mating suc- habitat, in which the water-agar and small rotting cess. First, pre-copulation ensures reproduction; pieces of potatoes offered hideaways, not all of second, it prevents other males from accessing to these 38 individuals were continuously visible at all a virgin female. Generally, ecological and behav- developmental stages. Therefore, figure 1 presents ioral data on astigmatid mites are poorly known a mosaic-like pattern of data from only those indi- (Evans 1992). Life cycle studies are thus required viduals that were visible for a whole duration of to understand the ecological role of mites in their a developmental stage including times of ecdysis. micro-habitats. The aim of the present paper is These were 1 – 3 specimens per stage, and its aver- to present some life cycle aspects of S. nipponensis. age duration is depicted in figure 1. The whole de- These data are based on specimens collected from velopmental durations of females and males of all USA, Louisiana. Furthermore, due to morphologi- 38 specimens are depicted in figure 2. cal differences between the type material of S. nip- ponensis from Japan (Tagami 2004) and the presently studied specimens of S. nipponensis from Louisiana, USA, sub-species level is discussed.

MATERIALS AND METHODS Specimens of S. nipponensis were sampled on Sar- racenia alata Wood, 1863 by S. Wirth in the Kisatchie National Forest (31°1’41. 64 N; 92°53’19.90 W), Alexandria (Louisiana, USA) in August 2007. The mites were cultured on 1.4 % water agar in Petri dishes of different sizes at about 24°C (± 3°C). The FIGURE 2: Individual whole development period of Sarracenio- water agar was prepared without sterilization, and pus nipponensis (USA) males and females, which were ob- the agar solution was casted into the Petri dishes served from the larval stage until adulthood at a temperature after being heated in a microwave. Two or three of 24°C (± 3°C) on daytime. On average, males need 6.2 days pieces of potatoes in form of cuboids (with a side (n = 16), females need 5.7 days (n = 22) to complete their de- velopment. length of about 1 cm and an altitude of 0.5 cm) were added to stimulate microorganism growth in Petri dishes with 5.5 cm in diameter, as food for the Not all individuals moulted into deutonymphs. mites. The potato pieces were regularly moistened Data about the deutonymphal development re- with water and once a month with beef heart solu- sulted from more or less freshly molted deu- tion instead of faucet water. Beef heart solution con- tonymphs, which were isolated from older cultures sists of faucet water, into which chunks of beef heart (9 cm in diameter), containing many mite individ- had been placed previously. Beefheart solution was uals, into smaller Petri dishes (5.5 cm in diameter), monthly newly prepared from small pieces (2 – 3 in which these deutonymphs could be observed in- cubes of meet with about 0.5 cm in diameter) of a dividually. Sex ratios were tested by placing mated frozen beaf heart that were put into water (a Petri females into Petri dishes (5.5 cm in diameter) and dish, 9 cm in diameter, nearly completely filled with rearing their offspring in the same dish until they water) and kept there for at least three days at room reached adulthood and also by observing entire cul- temperature (about 25°C). tures with many mite specimens. Isolated unmated Single larvae were isolated in small Petri dishes females produced no offspring; hence parthenogen- (5.5 cm in diameter) to determine the life cycle du- esis was not considered to be of importance in this

261 Krüger J. and Wirth S. species at our rearing conditions. The sex ratio of and tritonymph, Figure 1), Molting into deu- precopulated tritonymphs was determined by sep- tonymphs is facultative and usually did not hap- arating 18 couples in precopula into 5.5 cm Petri pen under the observed conditions. Instead proto- dishes each, after molting. nymphs molted directly into tritonymphs. For light microscopic preparations (Axioplan of At a temperature of 24°C (± 3°C), males and fe- Carl Zeiss, Germany), individuals of all stages (deu- males both developed in about 6 days (Figure 2) be- tonymphs, other nymphs and adults) were trans- ginning with the free-living larval stage. Adult fe- ferred into hydroxypropionic acid (98%); after a males lived for an average period of 46 days (38 – few hours, the ´s tissues were dissolved so 55; n = 4); males only 36 days (22 – 42; n = 4). that only the chitinous exoskeleton remained. Then The sex ratio was observed to be almost 1 : 3 mites were mounted on slides in "Berlese solution" when reared from eggs (6.2 males : 17.8 females). and after the medium solidified, the slides were Sex ratio data were also determined from culture sealed with glycel (a varnish). plates containing the offspring of many females. For scanning electron microscopy (SEM, Fei The sex-ratio observed then was of 1 : 2.5 (81 males Quanta 200) preparations, single individuals of : 204 females). These two ratios were quite similar. deutonymphs and adults were transferred into A dense aggregation of about 50 deutonymph ethanol (98%). They were then ultrasonically individuals was once observed; some had entered treated to remove dirt from their surfaces. Fixed quiescence towards the tritonymphal stage, some individuals were dried in 1, 1, 1, 3, 3, 3 were completing ecdysis, and others were still ac- -hexamethyldisiliazan and afterwards sputtered tive (Figure 3). with gold. The holotype and the paratypes of S. nipponensis (Japan) were studied and compared using the light- microscopy with the mites from Louisiana, USA. At least 50 slides with specimens of each, adult and deutonymph of S. nipponensis (Louisiana, USA) were used for these morphological studies. Images of the embedded objects were taken using a Nikon SC-17, an Axioplan light micro- scope and a Wild Heerbrugg M5A stereomicro- scope. Drawings were made using a camera lucida attachment on the Axiophot light microscope, and digitized with the aid of the drawing tablet Aiptek Hyperpen 12000U, the software Adobe Illustrator 10 and Adobe Photoshop 7.0.

RESULTS

Life cycle FIGURE 3: Aggregation of Sarraceniopus nipponensis (USA) deu- tonymphs during molting into tritonymphs. It took about 40 hours for the larva from hatch- ing the egg until the next ecdysis (Figure 1). From Mating and reproduction this point until the development of tritonymphs, mites needed another 82 hours, including two qui- Mate guarding or precopulation (Figure 5d) to mo- escence periods (20 – 26 hours between larva and nopolize female tritonymphs was observed regu- protonymph and 14 hours between protonymph larly in cultures of S. nipponensis from Louisiana.

262 Acarologia 51(2): 259–267 (2011)

FIGURE 4: Deutonymph of Sarraceniopus nipponensis (USA). a – dorsal view, b – ventral view, nomenclature of dorsal and ventral setae and conoids after Fashing & OConnor (1984). Legs I-IV. ϕ, ω, ω1, ω2, ω3, σ, σ1, σ2 are solenidia (setae as chemoreceptors). ih, im and ip are cupules (mechanoreceptors without hair-like structures on body surface). All other setae named in figure are mainly mechanoreceptors.

263 Krüger J. and Wirth S.

FIGURE 5: SEM pictures of Sarraceniopus nipponensis (USA). a – Dorsal view of the deutonymph, b – Detail of the cuticle’s surface, c – Ventral suckerplate, d – lightmicroscopic picture of two adult males trying to perform a precopulation.

Mate guarding was observed 8 hours before the detail. tritonymphs went into quiescence. Fifteen of the Reproduction in S. nipponensis was observed eighteen guarded tritonymphs that were examined only to occur after copulation with males. Tests after their molts were found to be females, and three with isolated virgin females showed no evidence were males, hence resulting in a sex ratio of 1:5 for parthenogenesis, as no eggs were produced. (males to females). Mated females produced 30 individuals on average within about 7 days (24 – 42; n = 5). Precopulated tritonymphs averaged almost the same idiosomal length as males (Figure 5d): 338 µm The mate guarding behavior was observed in (305 µm – 365 µm; n = 12): 346 µm (315 µm – 389 detail: males climbed onto the tritonymphal body µm; n = 5). Males apparently preferred tritonymphs from behind or from the side and finally came more or less their own size, even if further ex- to rest on the tritonymph’s dorsum. The whole periments would be needed to test this hypothe- procedure took about 15 minutes. The first legs sis. Indeed, we observed a larger variability in of the males clasped over the first pair of legs of tritonymphal sizes, but this was not measured in the tritonymph while legs III and IV of the male

264 Acarologia 51(2): 259–267 (2011) grasped the tritonymph’s body posteriorly behind morphological characters (Figures 4a, b, c), and its legs III and IV (Figure 5d). The second legs of therefore are interpreted by us to belong to the males often rested between tritonymphal legs II and same species, S. nipponensis. Slides with specimens III, but could also be found between tritonymphal from Louisiana, USA, are deposited in the collec- legs I and II. Legs I, III and IV of the male firmly tions of S. Wirth (Berlin, Germany), of J. Moser clasped the mate’s body, while the second legs (Pineville, Louisiana, USA) and additionally in the lay freely against the mate´s body surface and re- Museum für Naturkunde, Berlin (2 slides contain- mained mobile for defense against rival males. In ing several specimens, deutonymphs and adults, this way, the male guarded the tritonymph, which ZMB 48433, ZMB 48434). Specimens from both pop- was restrained directly underneath. The mating po- ulations could be compared using the holotype and sition on adult females was the same. Females were paratypes of S. nipponensis (slides) from Japan and observed to be copulated several times by differ- slides of mites from Louisiana, USA. They possess ent males. It is unknown, whether they are also the following synapomorphic characters of the deu- inseminated several times. In detail, two out of tonymph: the apodemes between leg II and III are three females observed for this specific behavior fused (Figure 4b). The sucker plate is reduced to a copulated and also produced offspring. After about vestigial structure and in particular the shape of its two weeks they were put into new dishes with new remnants, e.g. the small conoid-rests, is similar in males, where they copulated again. both species (Figures 4b, 5c). The deutonymph of The first legs of males are the thickest. They specimens from Japan (smooth dorsal surface) dif- were of special importance for climbing and clasp- fers from those from USA (Louisiana) in having a ing their mates. They additionally act as ’safeguard sculptured dorsal surface (Figures 4a, 5 a, b), being holders’. In case of disturbance during the mate also visible with the light microscope. guarding or finally during the copulation, these Two sclerotisations anterior to the dorsal legs never lost contact with the adult female or the hysterosoma in males occur in S. nipponensis tritonymph. During pre-copulation or the final mat- (Louisiana, LA), S. nipponensis (Japan) and S. dar- ing, males were never observed performing any lingtoniae. This character is interpreted as apomor- kind of active movements while riding the females; phy of the common stem species of these three instead they rested motionless on their mates. In ex- forms (Figure 6). Therefore S. darlingtoniae is hy- periments with water droplets that were dropped pothesized to be the sister-species of S. nipponensis, on pre-copulating and copulating couples males which is in consistence with the phylogenetic ar- were unable to stabilize their copulation position in gumentation of Fashing and OConnor (1984). An an upright position and slipped to the side of the apodeme that we herewith name p2z is distinctly female’s back. developed in the deutonymph; it seems to be of sys- The elongated hind legs of males were observed tematic relevance, and has never been emphasized to envelop the posterior end of adult females firmly, before (Figure 4b). It is interpreted as a plesiomor- but were found hanging limply on the agar sur- phic character, which presumably evolved in the face behind the mate when guarding very small fe- stem species of the genus Sarraceniopus. male tritonymphs (2 observations), or accidentally guarding against other males (2 observations). DISCUSSION Frequently, up to five males (5 males were ob- served once) were sitting on each other on a single The Sarraceniopus nipponensis population found on tritonymph during its molting (Figure 5d). S. alata in Louisiana, USA, may be a subspecies of S. nipponensis from Japan (Figure 6). Further inves- Phylogeny tigations are needed, whether morphological and Specimens of both populations from Louisiana, ecological differences refer to a subspecies’ level, or USA, and from Japan share important common just due to general variability within a same species.

265 Krüger J. and Wirth S.

FIGURE 6: Assumed phylogenetic relationships within Sarraceniopus.

To conclude, more specimens from Japan should be described for other histiostomatids such as Histios- included in further analyses. Ecological data of S. toma palustre Wirth, 2003 and H. feroniarum Dufour, nipponensis (Japan) are still missing. The compari- 1839 (Wirth 2004). son of ecological / behavioral characters may sug- Elongated hind legs of males were found in gest that both populations represent two different some histiostomatids such as Zwickia sp. (Fash- cryptic species. It could be also interesting to apply ing 2004b) belonging to the "pitcher-plant group". molecular markers to answer this latter question. These were used for active movements when the It is assumed that an active self-dispersal by mate was underneath, and fixed in this position by walking over short distances could be the preferred the forelegs. But the function of conspicuously elon- mechanism for dispersal of deutonymphs in S. nip- gated legs in males of S. nipponensis is still unclear, ponensis. Presumably, this allowed for the reduction because movement and control of the whole couple of the sucker plate components (Figures 4b, 5c). It by this on top sitting male using these legs to make is not known whether these sucker plates are still a contact to the ground as a possible function could able to attach the mites to other . It is not be observed. Generally, the conspicuously elon- thus possible that deutonymphs of S. nipponensis gated legs may just support a fixation of the male disperse through ambulatory activity rather than onto the female’s back during precopulation and phoresy, as described for S. darlingtoniae (Fashing copulation. 2004a). In three of 18 pre-copulations, males inadver- It was found that the male’s second legs have tently selected other males. Therefore, it can be as- additional functions during precopulation and cop- sumed that males can hardly differentiate the own ulation. It is adapted for clasping the female and gender from those of females during ecdysis. That defending the mate against rival males. This could was what Fashing (2004a) presumed for S. darling- be a typical histiostomatid feature, since it was also toniae. The sex ratio of 3 : 1 in favor of the females

266 Acarologia 51(2): 259–267 (2011) is conspicuous and differs from the ratio typically We are indebted to Jason Dunlop and J. Moser for found for other histiostomatids, which was mostly improving the English of the text. S. Wirth thanks 1:1 (Wirth 2004). the USDA Forestry-Service in Pineville/LA/USA Perhaps, the ability to differentiate tritonymphal and especially J. Moser that they invited him for a females from tritonymphal males is unnecessary research stay and that they supported him to get ac- within a population, since there is a threefold bet- cess to a Sarracenia alata area in Louisiana. We thank ter chance of encountering a female. G. raspotnig for comments to the manuscript, and S. Pre-copulation occurs many hours before Wirth thanks J. Dunlop for offering him a working tritonymphal ecdysis starts, which is different from place at the Museum für Naturkunde in Berlin. the behavior of males in Zwickia species. Males of Zwickia sp. seek already quiescent tritonymphs REFERENCES for guarding against competition from other males Evans, G.O. 1992 — Principles of Acarology — CAB In- (Fashing 2004b). The phenomenon of many males ternational, Wallingford (UK). 563pp. aggregating on single tritonymphs is not yet un- Fashing, N.J. 2004a — Biology of Sarraceniopus darlingto- derstood. There is no evidence that the male re- niae (Histiotomatidae: Astigmata), an obligatory in- leases his female tritonymph at any time during habitant of the fluid-filled pitchers of Darlingtnia cal- pre-copulation in such situations, or during the ifornica (Sarraceniaceae) — Phytophaga 14: 299-305. subsequent copulation. Direct fighting behavior Fashing, N.J. 2004b — Mate guarding in the genus Zwickia among aggregated males was not observed. Mass (Astigmata: Histiostomatidae) - a natatorial inhabitant of fluid-filled pitchers of Nepenthes (Nepentheaceae) copulations were observed in relatively old cultures — Phytophaga 14: 149-154. (i.e. more than three months old) with high popu- Fashing, N.J., Oconnor, B.M. 1984 — Sarraceniopus - a lation density, and never in younger cultures. Per- new genus for histiostomatid mites inhabiting the haps some sort of emergency causes this behavior pitchers of the Sarraceniaceae (Astigmata: Histios- at times when competition is extremely high. Com- tomatidae) — Int. J. Acarol. 10(4): 217-227. petition pressure presumably rises with increasing doi:10.1080/01647958408683380 numbers of males or decreasing number of females Hunter, P.E., Hunter, C.A. 1964 — A new Anoetus mite in the culture. from pitcher plants — Proceed. Entomol. Soc. Wash. 66(1): 39-46. In other species of Histiostomatidae, arrheno- Nesbitt, H. 1954 — A new mite, Zwickia gibsoni n. toky is commonly observed (unfertilized females sp., Fam. Anoetidae, from the pitchers of Sarrace- produce males). This differs from S. nipponensis, nia purpurea L. — Can. Entomol. 86(5): 193-197. as we show that fertilization is required to produce doi:10.4039/Ent86193-5 eggs. This does not mean that parthenogenesis gen- Tagami, K. 2004 — Sarraceniopus nipponensis sp. nov. and erally does not exist. It just means that a copu- S. hughesi (Astigmata, Histiostomatidae) from sarrace- niaceous pitchers bred in Japan — The Acarol. Soc. lation obviously is necessary for a production of Japan 13: 169-179. eggs afterwards. However, this phenomenon is not Wirth, S. 2004 — Phylogeny, biology and an isolate case among the family Histiostomatidae, character transformations of the Histios- since it was also observed in maritimum tomatidae (Acari, Astigmata). Disserta- Oudemans, 1914 (Wirth 2004). tion. FU Berlin. URL: (http://www.diss.fu- berlin.de/diss/receive/FUDISS_thesis_000000001415)

ACKNOWLEDGEMENTS COPYRIGHT We are grateful to W. Sudhaus and the work- Krüger and Wirth. Acarologia is under free li- ing group for providing suggestions and helpful cense. This open-access article is distributed under the criticism. We thank N. J. Fashing for reviewing terms of the Creative Commons-BY-NC-ND which per- the bachelor thesis, on which this paper is based, mits unrestricted non-commercial use, distribution, and and for some very helpful correspondence and K. reproduction in any medium, provided the original au- Tagami for sending type material of S. nipponensis. thor and source are credited.

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