To molt or not to molt: The dilemma of acquiring sexual maturity while losing durability and dispersal ability in deutonymphs of Parasitus fimetorum (Gamasida; Parasitidae)

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To molt or not to molt: The dilemma of acquiring sexual maturity while losing durability and dispersal ability in deutonymphs of Parasitus fimetorum (Gamasida; Parasitidae)

https://doi.org/10.21203/rs.3.rs-2101099/v1

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The dispersal from birthplace is an effective strategy to cope with unpredictable environmental change. In many animals, dispersal is carried out prior to mating and reproduction to avoid inbreeding. However, this also means that the dispersers have to find mate at the new habitat. Parasitus fimetorum (Acari; Parasitidae) is a free-living predatory mite inhabiting animal dung and manures. In such ephemeral habitats, the mites quickly develop from eggs to deutonymphs as an environmentally-tolerant, dispersal stage. After the dispersal to new habitat using dung beetles as phoretic hosts, deutonymphs have to make a decision; to molt to adult with losing the tolerance and migration ability or not to molt and continue migration. Here, using the molting-suppressed deutonymphs which were developed under isolated condition, I investigated whether food conditions (amount of rearing medium bearing nematodes as prey) and the encounter with conspecifics stimulate their molting. Drastic increase of the food resources induced molting of both sexes but the responses to the pairing treatment were different between sexes. Whereas female deutonymphs molted at the encounter with males irrespective of deutonymphs or adults, molting of male deutonymphs was induced only when paired with female deutonymphs (not adult females). Because the adult females are usually non-virgin and already started oviposition, the males seemed to discriminate against them. The decision about molting is understandable as a male mate choice, which is adaptive at the limited mating opportunities in the ephemeral environments.

Acari

Parasitus fimetorum

developmental arrest

decision making

molting condition

bet-hedging

mate choice

Many organisms live in changing environment (Levins 1968; Stearns 1992). The dispersal and migration from birthplace to another habitat is an effective way to cope with the environmental change (Ronce 2007). For many animals, such as insects living in ephemeral habitats, dispersal occurs before reproduction because the environment in which they are born quickly deteriorates and cannot sustain the next generation (Harrison 1980). Dispersal stage also has a great tolerance against unfavorable environmental conditions such as food shortage and desiccation. If the dispersers found favorable habitats, they change to a reproductive stage. However, the ability for dispersal and environmental tolerance are often lost once reproduction begins (Harrison 1980). For example, a cricket, Modicogryllus confirmatus shows wing dimorphism as the non-migratory short-winged morph and migratory long-winged morph (Tanaka and Suzuki 1998). After the migration, the long-winged morph absorbs the flight muscle and used the retrieved energy for egg production, and thus, inevitably loses flight ability (Tanaka and Suzuki 1998). Whereas the dispersal prior to reproduction has the benefit to avoid inbreeding, it entails potential risk that the disperser could not find mates. Therefore, these organisms should enter the reproductive stage only when they ensure favorable environmental condition such as abundant food and the existence of mate candidates.

Parasitus fimetorum (Berlese) (Acari; Gamasida; Parasitidae; Fig. 1) is a free-living predatory mite inhabiting animal dung and compost manure. The mites prey on fly eggs, nematodes and other small animals and quickly develop from egg to larva, protonymph and deutonymph within 3 days in the rapidly deteriorating habitats (Yasui 1993; Yasui 1997). Deutonymphs can tolerate starvation and desiccation for approximately a month, during which they disperse to new habitats on their own legs or via phoresy on dung beetles (Aphodiinae) (Yasui 1993; Yasui 1997). At the destination, deutonymphs molt to adult and lose the migrating ability and tolerance (adult longevity is approximately one week; Yasui 1993; Yasui 1997). In another dung-inhabiting mite Macrocheles muscaedomesticae (Scopoli) (Gamasida; Macrochelidae), having haplo-diploid sex determination system, females copulate just after adult molting with a male that has been guarding them for several hours (precopulatory mate guarding; (Yasui 1988). Males die at the birthplace and adult females disperse by phoresy on house flies (Muscidae). In Macrocheles, only adult females have the durability and migrating ability so that the developmental arrest in immature stage does not exist. Thus, Macrocheles females usually finish copulation before dispersal and even if they could not find mates, virgin females can produce male offspring by arrhenotokous parthenogenesis. In contrast, Parasitus has diploid-diploid genetic system and conducts only bisexual reproduction (no parthenogenesis) (Ito 1973; Ito 1978; Yasui 1993; Yasui 1997), meaning that they have to find mates after dispersal. Therefore, I expected that P. fimetorum deutonymphs molt to adult only when they encountered mate candidates under promising food condition. If deutonymphs molt to adult without satisfying these conditions, the emerged adults must die without reproduction. Herein arises a dilemma to molt or not to molt.

In this study, I examined whether P. fimetorum deutonymphs that halted development under isolated rearing conditions molt or not when (1) food availability is drastically improved and when (2) they encountered another conspecific individual (same or different sex, deutonymph or adult).

Mites

The population of P. fimetorum used in this study was collected from a compost pile in Faculty of Agriculture, Kagawa University, Shikoku-island, Japan (N34°16' 35.86", E134°07' 29.58", altitude 24 m) in April 2002 and established by approximately 100 wild-born deutonymphs. The population was maintained on an artificial medium, which consisted of moistened wheat bran, rice husks and compound food for rabbits, in a plastic container (125 mm in diameter, 50 mm in depth, the lid with an opening of 40 × 50 mm and covered by fine polyester mesh; the window size was changed by vinyl taping according to the moisture inside) and kept in an incubator at 25 ± 1°C, 16L8D. The free-living saprophagous nematodes (Rhabditida) were allowed to reproduce in this medium as prey for the mites. Several containers, each containing hundreds of mites were maintained as stock cultures. The experiments were carried out at Laboratory of Entomology, Faculty of Agriculture, Kagawa University from April 2002 to January 2003.

To collect newly laid eggs, one or two pair(s) of adult were introduced from the stock culture to a plastic container (36 mm in diameter, 20 mm in depth, the lid with an opening of 10 mm in diameter covered by fine polyester mesh) equipping plaster-charcoal substratum on the bottom. A small piece of moistened rabbit food containing nematodes was fed on a 10 × 10 mm aluminum foil. Females prefer to lay eggs on loosened absorbent cotton fiber. The eggs were isolated in a glass vial (18 mm in diameter, 45 mm in depth, equipping plaster-charcoal substratum and a food piece) and incubated. Every day, I checked the vials and obtained the deutonymphs of both sexes that molted within the past 24 hours and had not yet encountered other individuals, referring to as 0-day old solitary deutonymphs. They were sexed on the basis of morphological differences on the upper ventral surface of idiosoma (the location of genital opening in adult males; Fig. 2).

Experiment 1: Molting of solitary deutonymphs when food conditions were drastically improved

I investigated whether the drastic increase of food induced the molting of deutonymphs even if they did not contact with other individuals. An individual which was isolated at the egg stage was fed fresh rabbit food containing nematodes on a 5 × 5 mm aluminum foil in the glass vial. This food amount was sufficient for the development of an individual but not for offspring population. In this rearing condition (control), the food was renewed every day, and the molting to adult was checked for 10 days. I made 3 treatment groups. At the various timing of ontogeny (protonymphs ca. 2 days before molting to deutonymph, 3-day old and 6-day old deutonymphs), a mite was transferred from the control group into a plastic container (36 mm in diameter, 20 mm in depth, the lid with a meshed window) full of fresh rearing medium. A piece of the nematode bearing rabbit food was added to the container, resulting in explosive increase of nematodes (approximately as 1500 times many as the control). Because of the difficulty to find a single mite in the rearing medium, I intermittently checked the molting to adult for two or three weeks after the start of the experiment.

Experiment 2: Molting of deutonymphs at the pairing with various counterparts

The deutonymphs that did not molt for 10 days in the moderate food condition (i.e., control in experiment 1) were considered to enter the developmental arrest. I made a pair of individuals in a glass vial with plaster-charcoal substratum in the following combinations; 1) two arresting deutonymphs of the same sex, 2) two arresting deutonymphs of the different sex and 3) an arresting deutonymph + an adult of the opposite sex. As a control, deutonymphs were kept in solitary conditions (this was a replicate of control in experiment 1 but continued for longer period). The mites were kept in the moderate food condition. I checked all deutonymphs every day until their death. If one of the pairing partners died within 4 days from the start of experiment, another individual was excluded from the analyses.

For statistical analyses, JMP ver. 10.0.2 for Windows was used.

Experiment 1: The effects of food conditions on molting

Fig. 3 shows the cumulative molting rate of solitary deutonymphs. Under moderate nutrient conditions (control), solitary deutonymphs exhibited low molting rate (Fig. 3d). The cumulative molting rate on the 10th day was 3-times higher in males (26/99) than in females (10/135). Survival analysis (Cox proportional hazards model) detected a highly-significant difference between sexes (no. parameters = 1, d. f. = 1, likelihood ratio Chi-square = 15.3555, P < 0.0001). No individuals died during 10 days.

However, when food resources were drastically increased, the solitary individuals quickly respond (Fig. 3a~c). More than 50% of individuals molted to adult within a few days. Males show the higher molting rate than females. Survival analysis (Cox proportional hazards model) detected a highly-significant difference among the four groups (Fig. 3) (no. parameters = 3, d. f. = 3, likelihood ratio Chi-square = 38.4473, P < 0.0001) and between sexes (no. parameters = 1, d. f. = 1, likelihood ratio Chi-square = 21.3086, P < 0.0001). Even if the control samples (n = 234, 84% of all samples) are excluded from analysis, the differences are still significant among three treatments (Fig. 3a~c) (no. parameters = 2, d. f. = 2, likelihood ratio Chi-square = 7.1877, P = 0.0275) and between sexes (no. parameters = 1, d. f. = 1, likelihood ratio Chi-square = 7.1954, P = 0.0073).

Experiment 2: The effect of pairing on molting

Fig. 4 shows the cumulative molting rate of a deutonymph (DN) paired with another individual (two individuals in a vial). In the pairs of two deutonymphs, molting of both individuals were tracked. Thus, “DN♀ + DN♀, n = 38” (Fig. 4a) means 16 pairs of female deutonymphs (16 vials) were investigated. In the pairs of a deutonymph and an adult (A), only deutonymphs are checked so that “DN♀ + A♂, n = 16” (Fig. 4c) means that 16 pairs (16 vials) were examined.

When paired with a deutonymph of the same sex (Fig. 4a), approximately 50 % of female deutonymphs and 40 % of male deutonymphs finally molted within a month. Survival analysis (Cox proportional hazards model) detected no significant difference in the molting rate between sexes (no. parameters = 1, d. f. = 1, likelihood ratio Chi-square = 0.8426, P = 0.3587).

Out of 18 heterosexual pairs of deutonymphs (Fig. 4b), three females molted within a day but died before male molting. Males of these pairs were excluded from the analysis, so that the male sample size decreased to 15. Females and males in almost all pairs molted to adult within a few days. The molting rate of females was slightly higher than that of males but survival analysis (Cox proportional hazards model) detected no significant difference between sexes (no. parameters = 1, d. f. = 1, likelihood ratio Chi-square = 0.3631, P = 0.5468).

When paired with an adult of the opposite sex (Fig. 4c), female deutonymphs immediately molted but male deutonymphs remained not molted. Survival analysis (Cox proportional hazards model) detected a highly-significant difference between sexes (no. parameters = 1, d. f. = 1, likelihood ratio Chi-square = 16.7439, P < 0.0001). The molting rate in these males was as low as that in the solitary condition (ca. 35%; Fig. 4d).

The solitary deutonymphs (control; Fig. 4d) survived for longer than a month without molting. The cumulative molting rate at the end was ca. 35% for both sexes. Survival analysis (Cox proportional hazards model) detected no significant difference between sexes (no. parameters = 1, d. f. = 1, likelihood ratio Chi-square = 0.1048, P = 0.7460).

In conditions where deutonymph was alone, males tended to molt earlier than females (protandry; Figs. 3, 4d), whereas in conditions where deutonymph was combined with other individuals, females tended to molt earlier than males (protogyny; Fig. 4a-c).

In summary, the pairwise comparison of the maximum cumulative molting rate in all 6 combinations among 4 treatments using Fisher’s exact probability test with Holm-Bonferroni sequential correction (Fig. 5) detects a sex difference in the response. All female deutonymphs molted at the combinations with males (irrespective of stages) but in the pair with another female deutonymph, they showed low molting rate that did not differ from solitary condition (Fig. 5a). On the other hand, the molting rate of male deutonymphs were still low at the pairing with female adults (Fig. 5b).

This study clarified that (1) the drastic increase of food resources stimulates the dormant deutonymphs of P. fimetorum to molt and (2) the cohabitation with another individual, especially of the opposite sex, induces the molting of the dormant deutonymphs. In addition, I established a sexing method of living deutonymphs of this species. Hereafter, I discuss these findings in detail.

Adaptive molting decision

When deutonymphs molt to adult, the tolerance against harsh environments (starvation, desiccation etc.) is lost (Ito 1978; Yasui 1993). Even if a few individuals that were settled in a new habitat successfully reproduce after molting, their offspring also have to reach deutonymphal stage for acquiring the tolerance and dispersal ability to the next habitats. Thus, abundant food resources sufficient for the development of offspring population (not for the immigrated individuals) are required as a molting condition. The low molting rate in Fig. 3d and high molting rate in Fig. 3a~c are understandable for this reason. In experiment 1, the individuals entering promising food environments quickly developed to adult without contact with other individuals. This may be an artefact in the experimental condition. Such a good patch containing huge food resource would also accept many other immigrants in nature, so that the molted individuals can find mate sooner or later. Fast development (higher intrinsic rate of natural increase; Pianka 1970) is advantageous in an expanding population. Thus, the drastic improvement of nutritional condition may stimulate solitary deutonymphs to molt.

However, under moderate food condition, which was sufficient for development of a few mites, deutonymphs molted only when they ensure potential mates, especially deutonymphs of the other sex (Fig. 4b). Interestingly, whereas female deutonymphs did not discriminate between male adults and male deutonymphs (in the form of molting decision), male deutonymphs discriminate against female adults (Fig. 4c).

Male choice

Molting of deutonymphs at the pairing can be interpreted as a kind of mate choice. The present study shows that male deutonymphs of P. fimetorum is more choosy than female deutonymphs. Sexual selection (Andersson 1994; Darwin 1871) works through intra-sexual competition and inter-sexual mate choice. Because of the inter-sexual difference in potential reproductive rate (Clutton-Brock and Vincent 1991) due to the difference in gamete production ability between sexes, the competing sex is usually male and the choosing sex is usually females in most animals. However, if mating opportunity is limited and if there is great variation in female quality, males cannot fully achieve their potential fertilization ability and thus, choose mates (Ball and Parker 2007; Johnson and Hubbell 1984; Schwagmeyer and Parker 1990). For example, males of M. muscaedomesticae choose young virgin females because only the first male to mate with a female can fertilize her eggs (i.e., perfect first-male sperm precedence; Yasui 1988; Yasui 1996). For male deutonymphs of P. fimetorum, adult females may be low-quality mate candidates because in most cases they would have already mated with other males and laid certain amount of eggs, meaning the small residual reproductive value (Yasui 1996; Yasui 1997). Short longevity (ca. 1 week) of adult males limits their mating opportunity. In contrast, for female deutonymphs, males irrespective of adult or deutonymph could be appropriate mates. Therefore, only male deutonymphs exhibit mate choice via molting decision.

How to sex deutonymphs – a break through

The suspension of molting (a kind of dormancy) of deutonymphs and its resuming conditions, especially the contact to the individuals of different sex, was reported in other species of Parasitidae (Ito 1973; Ito 1976; Ito 1978; Nehring and Müller 2009). However, the difficulty of sexing at deutonymphal stage has made the pairing experiment controlling the sex combinations difficult. Ito (1973, 1976, 1978) sexed deutonymphs of P. gregarious based on carmine-color pigmentation of ventral surface, which appears only in two or more weeks old males. This is too late to detect the decision making of young deutonymphs. Nehring and Müller (2009) did not sex deutonymphs of Poecilochirus carabi and instead, indirectly estimated the probability that the pairing group of deutonymphs contains both sexes under the assumption of 1:1 sex ratio, which is quite frustrating. Based on the newly-found morphological sex difference (Fig. 2), which may be common among paraistid species, the present study enabled to clarify the molting decision of young deutonymphs.

Remaining issues

How do they recognize the developmental stage and sex of the encountering conspecifics? Ito (1976) showed that direct contact with heterosexual counterparts induced the molting of P. gregarious deutonymphs and suggested that some tactile and/or chemical stimulation might be involved. Such proximate factors are plausible in P. fimetorum. If they also recognize and discriminate kin or non-kin, the mate choice could function as inbreeding avoidance. If the deutonymphs descended from a single mother disperse to multiple patches by riding on the different dung beetles, the mother’s lineage would avoid from extinction unless all patches simultaneously suffer bad conditions. Thus, reproduction after dispersal could work as spatial risk-spreading or metapopulation bet-hedging (Hopper 1999; Yasui 2022). Nehring and Müller (2009) reported that male deutonymphs of Poecilochirus carabi do not molt if adult males exist nearby, to avoid cannibalism by adult (as male-male competition) at the vulnerable period immediately after molting. If this is also the case in P. fimetorum, it may explain the protandry in the solitary condition but the protogyny in the cohabited conditions. The male would probably molt while his rival is absent. These untested possibilities are remained for future researches.

Acknowledgements

M. Ogawa assisted to collect experimental data. K. Matsumura made valuable comments on the draft and provided statistical tests.

Funding

This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports Science and Technology (MEXT) of Japan to Yasui (nos. 26440241 and 19K06839).

Data availability

The datasets supporting this article have been uploaded as part of the electronic supplementary material.

Confict of interest

I declare that I have no competing interests with other persons.

Ethics approval

The mites (P. fimetorum) used in this study are invertebrates and therefore have not been subjected to animal ethics review. The study was conducted in a manner that avoided or minimized discomfort or distress to the laboratory animals, and efforts were made to ensure that the animals did not suffer unnecessarily at any stage of the experiment. The laboratory population of P. fimetorum used in this study have maintained at Kagawa University. This population has been maintained on the artificial medium in which nematodes as their natural prey reproduce. We reared this population at 25 °C, which is comfortable condition for this mite. All animals in the study were handled carefully.

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No competing interests reported.

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To molt or not to molt: The dilemma of acquiring sexual maturity while losing durability and dispersal ability in deutonymphs of Parasitus fimetorum (Gamasida; Parasitidae)

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