Experimental and Applied Acarology 26: 79–86, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.

Mating and fecundity of Dermatophagoides farinae ANDREA ALEXANDER, NDATE FALL and LARRY ARLIAN* Department of Biological Sciences, Wright State University, Dayton, OH 45435-0001, USA; *Author for correspondence (e-mail: [email protected]; phone: (937) 775-2568; fax: (937) 775-3320) Received 12 September 2001; accepted in revised form 1 March 2002

Key words: Dermatophagoides farinae, Fecundity, House dust mite, Multiple matings Abstract. Studies of the life cycle of cultured Dermatophagoides farinae found that after an initial mating D. farinae females lived for 63.3 ± 64.6 (SD) d after their egg production period ended (Arlian and Dippold 1996). The long period after cessation of egg production for D. farinae suggested D. farinae females could mate multiple times and produce eggs continuously for a longer period. The purpose of this study was to determine if female D. farinae could mate at least two times, and subsequently increase the production of viable eggs over a longer period of time compared to a single mating. Female D. farinae were randomly selected from thriving cultures and isolated in cages. When the females had ceased to lay eggs a male was added to the cage. Fifty-seven percent of the isolated females mated again and produced a second batch of viable eggs. In natural or culture populations, females have continuous availability of males. Therefore, in another experiment, females that emerged from the tritonymphal stages were continuously exposed to fresh males and fecundity and lengths of the reproductive and post reproductive periods were determined. These females had a 11 d longer reproductive period and produced 30.7% more eggs compared to females that only mated one time after they emerged from the tritonymphal stage. However, the post reproductive period was still long (58.6 ± 11.4 [SE] d) the significance of which is not clear. In conclusion, this study revealed that D. farinae females are capable of more than one successful mating that results in increased egg production compared to that of a single mating. It is likely that females mate multiple times in natural and culture populations. It was observed that females actively attract males during the reproductive period but not afterward even though she continues to live a long time.

The house dust mite, Dermatophagoides farinae Hughes, is the source of multiple potent allergens in house dust that stimulate allergic responses in a high percentage of house dust-sensitive individuals. This species commonly occurs in homes worldwide that are located in humid climates. D. farinae has a high biotic potential and natural populations in homes during humid summer months can increase exponentially when food and moist air are not limiting (Arlian et al. 1982). Population growth rates can be 17%/wk in laboratory cultures (Arlian et al. 1998a). Recommendations for eradication of these mites in homes include reducing indoor relative humidity, washing bedding and clothing in hot water (>55 °C) and applying acaricides (McDonald and Tovey 1992; Dietemann et al. 1993; Arlian et al. (1999, 2001); Arlian and Platts-Mills 2001). A greater understanding of the biology of D. farinae may lead to development of new strategies for control of these mites in homes.

80 Laboratory studies showed that D. farinae developed well at 23 and 30 °C, but most eggs did not develop to adults at 16 and 35 °C (Arlian and Dippold 1996). After an initial mating following eclusion to adult at 23 °C and 75% RH, the reproductive period for D. farinae was 34.0 ± 10.7 (SD) d, and they produced 2.0 ± 0.4 (SD) eggs/d (Arlian and Dippold 1996). The average time from emergence to the cessation of egg production was 36.6 ± 10.7 (SD) d but females lived 100.4 ± 59.8 (SD) d. Therefore, D. farinae females continued to live for 63.3 ± 64.6 (SD) d even though no eggs were produced. The long period after egg production for D. farinae suggested that it might be possible for females to mate multiple times and thus produce more eggs over a longer period of time. The purpose of this study was to determine if D. farinae could mate multiple times and subsequently increase production of viable eggs over a longer period of time compared to the number produced following a single mating.

Methods Dermatophagoides farinae mites were taken from thriving laboratory cultures maintained as previously described (Arlian and Dippold 1996). Egg production following second or subsequent mating One hundred and five females were selected randomly from four thriving laboratory cultures and each female was caged separately in 4 mm i.d. by 25 mm glass tubes closed at the ends with 35␮m nylon mesh held in place by Teflon (DuPont, Wilmington, DE) washers as previously described (Arlian et al. 1990). Culture medium was placed in each cage initially and added when needed throughout the study. The cages with mites were placed in petri dishes, which were kept in ventilated humidity chambers maintained at 75% RH and room temperature (20 – 22 °C). The cages were observed approximately 3 times a week using a stereomicroscope and the number of eggs produced were recorded and removed from the cage via probe. When the female had ceased to lay eggs for > 10 consecutive days a male was added. If the female did not show subsequent egg production during a period of ⭓ 10 d after the first male had been added, a new male was added. The experiment ended after egg production had ceased for greater than 20 d. Viability of eggs A separate study was conducted to test the viability of the eggs that were produced during the second reproduction period. Fifty D. farinae females were selected from two thriving laboratory cultures and caged in glass tubes as described above. As before, culture medium was placed in each cage initially and added when needed throughout the study and cages were held at conditions described above. The cages were examined using a stereomicroscope approximately 3 times a week and the

81 eggs produced were removed via probe. It was noted whether or not eggs had been produced. When the female had ceased laying eggs for > 10 consecutive days a male was added. If the female did not start producing eggs in ⭓ 10 d after the first male had been added a new male was added. If the female showed a subsequent egg producing period then the number of eggs produced per day was recorded and the location of each egg in the glass cage marked in order to keep track of the egg. The eggs were observed and when the larva emerged it was removed from the cage. Since the reported average incubation times at these test conditions was 10.1 ± 0.8 (SD) and 13.8 ± 1.2 (SD) d (Arlian and Dippold 1996; Arlian et al. 1998b), eggs that did not hatch in 33.5 ± 2.57 (SE) d (range 18 to 59) were considered not viable. The experiment ended after egg production by each female had ceased for ⭓ 20 d and all of the eggs that were produced had hatched or were monitored for 33.5 ± 2.57 (SE) d. Effects of continuous exposure to males A separate experiment was conducted to determine the effect continuous availability of males would have on female fecundity, duration of the reproductive period and longevity. Fifty D. farinae tritonymphs were selected from two thriving laboratory cultures and caged in glass tubes as described above. Culture medium was placed in each cage initially and added when needed throughout the study. The cages with mites were held as described previously. The cages were examined daily and emergence from tritonymph to the adult stage (male or female) was recorded. If males emerged from the tritonymphs they were removed from the study. If females emerged, two males (that were taken from thriving laboratory cultures of D. farinae) were added to the tube. Males were replaced every 10 d with two fresh males. The numbers of eggs produced were recorded and removed daily. The experiment ended when all of the females had died. As a control, isolated virgin males and females that had emerged from tritonymphs were held in a similar manner in the absence of the opposite sex until they expired. Also as a control, females were held without males for 49 days and then males were added to determine if the aged females would mate and produce eggs. Data The data are reported as means and standard errors. Statistical analysis was performed using a two-sample t-test assuming unequal variances.

82 Table 1. Comparative longevity and reproductive period data for mated, remated, and frequently mated D. farinae females. Parameter

Single Mating* (n=41)

Second Mating (n=35)

Continuous Mating** (n=27)

Female longevity, days Reproductive period,

100.4 ± 10.1 34.0 ± 1.8

18.1 ± 1.4

112.2 ± 10.9 44.8 ± 3.1

days No. eggs per day per

2.0 ± 0.07

0.9 ± 0.1

2.2 ± 0.06

female (for period) Mean no. of eggs

65.5 ± 2.9

15.3 ± 1.6

94.7 ± 6.3

produced * Data from previous life cycle study (Arlian and Dippold 1996) ** Unmated females lived 195.7 ± 5.5 days ** Unmated males lived 155.2 ± 4.2 days

Results Egg production following second mating Of the 105 females originally confined to cages, 44 were removed from the study. Of the 44 females discarded, 12 were killed during handling, 5 received a male too soon after egg production ceased (< 10 d), 2 never produced eggs throughout the study (possibly because the females were infertile or had already completed egg production), and 25 died before a male was added. These 25 may have died from natural causes because they were already at the end of their egg producing period and had reached their natural longevity. Therefore, these results were based on the 61 females that produced eggs. The mean number of days after the last egg was laid until the day a male was paired with the 61 females was 20.8 ± 0.9 d (range 11 – 38 d). After the introduction of males 35 of the 61 females (57.4%) produced eggs again. The average number of eggs produced by the 35 females during the second reproductive period was 15.3 ± 1.6 (range 1 – 58) (Table 1). The mean number of eggs produced per day during the second reproductive period was 0.9 ± 0.1. The average length of the second reproductive period was 18.1 ± 1.4 d with a range of 2 to 42 d. Viability of eggs Of the 50 females originally confined to cages, 22 were removed from the study. Of the 22 removed, 5 were killed during handling, 1 received a male too soon after egg production ceased (< 10 d), and 16 died before a male was added. Therefore, these results were based on the 28 females that produced eggs. After the introduction of males, 15 of the 28 females (53.6%) produced eggs (range 1 – 37) a second time. The 15 females produced a total of 216 eggs. After incubation, larvae emerged from 149 of the 216 eggs. Therefore, 69.0% of the eggs

83 produced during the second reproductive period were viable. The remaining eggs did not hatch after 33.5 ± 2.6 d (range 18 – 59) of incubation and were therefore considered not viable. Effects of continuous exposure to males Of the 50 tritonymphs originally confined to cages, 20 males emerged, and were removed from the study. Of the 30 females that emerged in the confined cages, 2 were killed during handling and 1 was lost. Therefore, these results were based on 27 females that emerged from the tritonymphal stage. Following the initial introduction of males, all 27 females produced eggs. The number of eggs produced by individual females ranged from 32 – 136. The mean number of days the 27 females survived was 112.2 ± 10.9 d (range 30 – 232) which was significantly different (p<0.005) compared to the longevity of unmated females. Females that were continuously exposed to males had an average reproductive period of 44.8 ± 3.1 d (range 13 – 73) (Table 1). The average number of egg producing days (from the first day an egg was produced until the last day an egg was produced) was 39.5 ± 2.7 d (range 13 – 69). Mean total number of eggs produced per each female was 94.7 ± 6.3 eggs (range 32 – 145). During the reproductive period, the average number of eggs produced per egg-laying day for each female was 2.4 ± 0.05 (range 1.9 – 2.9). The number of eggs produced per female per day was 0 to 7. The average post reproductive period was 58.6 ± 11.4 d (range 0 – 213). The longevities of unmated females and males were 195.7 ± 5.5 (n=15) and 155.2 ± 4.2 (n=15) d, respectively.

Discussion Multiple matings among astigmatid mites are common (Pallai and Winston 1969; Fashing 1975; Griffiths and Boczek 1977; Boczek and Griffiths 1979; Radwan and Siva-Jothy 1996). It is estimated that Acarus siro mates at least once a day while Caloglyphus berlesei and Rhizoglyphus robini mate once/h (Boczek and Griffiths 1979; Radwan 1991; Radwan and Siva-Jothy 1996; Radwan and Rysinska 1999). Until now, multiple matings among the Dermatophagiodes spp. have not been investigated. A previous study found that female D. farinae mate soon after they emerge from the tritonymphal state (Arlian and Dippold 1996). In thriving laboratory cultures, females are often observed to be mounted multiple times by males in a copulatory position or have males pursuing them. While this behavior suggests multiple or remating it does not prove that copulation and insemination with viable sperm occurs multiple times. Our first two experiments of this study were designed such that males were not available to females until after a period in which no eggs were produced. This was

84 the only way to determine that a second successful insemination that resulted in production of viable eggs was possible. These experiments revealed that after an initial mating and egg producing period D. farinae females could be mated again and then produce a second small batch of fertile eggs. This second reproductive period was short (18 d) compared to the first (34 d). Regardless, this was direct evidence that D. farinae was capable of successful multiple matings. Therefore, multiple matings likely occur in natural populations. It is not clear why females stopped laying eggs after the first mating and then could resume again after a second mating. Presumably, the sperm supply from the first mating was depleted and egg production stopped although we have no direct evidence for this. These results do suggest that factors from the male prime and induce egg production by the female. Apparently, the presence of the male and/or insemination and insemination products induced this response. In their natural environment or in cultures females have continuous availability of males and multiple matings likely occur during the reproductive life of the female. Therefore, the third experiment was performed to determine if the females reproductive potential was increased by multiple matings when continuously exposed to males. Under these conditions, it was not possible to determine if or how many multiple successful matings occurred. However, it was possible to monitor egg production per day, length of the egg laying period and the longevity of females in the continual presence of males and then compare these results to females that mated once and females remated after the first reproductive period. We found that following emergence from the tritonymph, females with continuous exposure to males, produced 44.6% more eggs than the total number of eggs produced following a single mating and about 17% more than produced by a first and second mating combined. The egg producing period was increased by about 11 d or 32% compared to that of a single mating but it was about 7 d less than that for a combined first and second interrupted reproductive periods. Both the total numbers of eggs and the oviposition periods of females continuously exposed to males were statistically different (p<0.005) compared to those females only mated a single time. Thus, continuous multiple matings did increase the egg producing potential but this occurred over a shorter time compared to two interrupted sequential matings. These findings indirectly indicate that in natural or cultured populations two or more successful inseminations likely occur. The controls for this experiment showed that like other astigmatid mites the unmated or less frequently mated D. farinae females lived longer than those that mated repeatedly (Fashing 1975; Rivard 1959; Pallai and Winston 1969). We also found that females that were deprived of a male for 49 days could mate and produce eggs later in their life. We did observe that when males were continuously present during the entire egg laying period males constantly accompanied females and mounted them in a copulating position many times. When egg production ended males no longer showed any interest in the females and were seldom seen in the vicinity of the females. This suggested that during the reproductive period females produced a sex attractant that was not present once the female had expended her reproductive potential. A similar phenomenon has been observed for Naiadacarus arboricola an aquatic

85 astigmatid mite that lives in tree-holes (Fashing 1975). Regardless of whether females of D. farinae had males available once or continuously, females in both situations exhibited a long post reproductive period, as previously reported (Arlian and Dippold 1996). The significance of the long post reproductive period is not clear. Clearly, our studies show that multiple matings likely occur and increase the fecundity, population growth, and biotic potential of D. farinae. The full significance of multiple mating by this species is not clear. As discussed by Burpee and Sakalik (1993), multiple matings may ensure that the viable sperm reservoir in the seminal receptacle is not depleted before the female reaches the limits of her reproductive potential. It also creates competition between sperm of different males and thus increases the genetic fitness and diversity of offspring (Radwan and Witalinski 1991).

Acknowledgements This research was partially supported by the U. S. Environmental Protection Agency Grant No. R 825250-01-0

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