Ecological Entomology (1995) 20. 230-236

Sexually transmitted disease in a promiscuous , bipunctata

GREGORY D. D. HURST, ROSIE G. SHARPE," ANGELA H. BROOMFIELD, LINDA E. WALKER, TAMSIN M. 0. MAJERUS, ILIA A. ZAKHAROVt and M I CH A E L E . N . M A J E RU S Department of Genetics, University of Cambridge, *Department of Genetics, University of Leeds, and 'N. 1. Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia

Abstract. 1. Sexually transmitted diseases (STDs) have rarely been reported in and other invertebrates. The majority of those reported involve organisms where sexual transmission is augmented by either vertical (i.e. inherited) trans- mission, or horizontal transmission, independent of host sexual activity. 2. We here demonstrate the existence of an STD in the coccinellid Adufiu bipunctuta. This species bears a parasitic mite of the genus Coccipolipus. We show that, like many other podapolipid mites, this mite is transmitted between host individuals at a high rate during copulation. It also appears to be transmitted at a low rate between non-copulating individuals. 3. We show that infected female A.bipunctatu produced at a reduced rate, and that the eggs produced by infected females have highly decreased viability. However, no effect of infection upon host longevity was observed. 4. The results are discussed in relation to the incidences of sexually transmitted disease in invertebrates in general, the causes of disease symptoms, and the importance of this disease in the evolution of A.bipunctutu.

Key words. STD, venereal disease, sexually transmitted disease, Coccipolipus, Adulia bipuncrura, , inherited parasites. lntroductlon mating, and causes a discernable pathology in its host (Marti et af., 1990; Simmons & Rogers, 1990). In the Sexually transmitted diseases (STDs) are known to have a Indian rhinoceros beetle, Orcytes rhinoceros, a baculovirus considerable demographic impact on humans and other is transmitted between partners, and reduces host longevity promiscuous birds and mammals (Anderson & May, 1991; (of both male and female) and progenic survivorship (of Smith & Dobson, 1992; Sheldon, 1993). However, the the female) (Zelazny, 1972, 1973a, b, 1976). The status of number of well-documented incidences in invertebrates is other examples of STDs in insects has not yet been fully remarkably small (Smith & Dobson, 1992). One definite established. Although many insects have been suspected of example is in the Dungeness crab, Cancer magister bearing sexually transmitted viruses, and protozoa (Wickham et al., 1984). In this case a nemertean worm, which produce diseases, in no case is there good evidence Carcirionemertes errans, consumes the eggs of the female both that this sexual transmission occurs in the wild, and host, before producing a planktonic larval stage (Wickham, that the organism produces a fitness loss to its host. For 1980). Larval nemerteans alighting upon a male individual instance, one of the most frequently cited cases of venereal (which produce no eggs to consume) transfer to the transfer is that of Lacrosse virus from male to female female during copulation. Two cases are documented in Aedes afbopictus (Thompson & Beaty, 1977); however, insects. In the fall army-worm, Spodopfera frugiperda, an experiments have failed to show any effect on the fitness ectoparasitic nematode transfers between partners during of venereally infected individuals (Patrican & DeFoliart, 1985; Patrican et a/., 1985, but see Grimstad el a/., 1980). Corrcspondcncc: Dr G. D. D. Hunt. Dcpartmcnt of Gcnctics, One other group of parasites which has been proposed Downing Strcct. Cambridge CB2 DEH. as being the aetiologic agent of an STD are the mites of the

230 Sexually transmitted disease in A. bipunctata 23 1 family Podapolipidae. Podapolipid mites are found on sex). All ladybirds were marked with a spot of coloured Coleoptera, Orthoptera, Hymenoptera and Dictyoptera. TippexTMon their elytra in order to distinguish individuals. They are characterized by having sessile adults which The ladybirds were observed for 1 h under a bright fluor- feed on the adult host, and motile larvae. These larvae escent light (22 W), and all contact between individuals have been either directly observed moving between hosts was recorded. during copulation (Podapolipoides grassi on the locust After the hour had elapsed, donor sex individuals which Chortoicetes terminifera: Gauchat, 1972; Coccipolipus were not mating were removed, and kept singly in petri epilachnae on the coccinellid beetle Epilachna varivestris: dishes. Other individuals of the donor sex were removed Schroder, 1981; Eutarsopolipus regenfussi on the carabid following termination of mating. This prevented unrecorded beetle Chlaenius pennsylvanicus: Husband & Swihart, matings occurring after the end of the observation period. 1986), or such movement has been inferred from their When all donor sex individuals had been removed, the position on the organism (Ovacarus clivinae and 0.peelei recipient sex individuals were removed to individual petri are found attached to the vaginal membrane of their hosts, dishes. the Clivinia impressifrans and Pasimachus elongatus The number of larval mites found on each of the ladybirds respectively: Stannard & Vaishampayan, 1971; Husband, was recorded for each ladybird in the experiment after 1974). copulation. The experiment was performed ten times with The podapolipid mites certainly appear parasitic. How- the male as the donor sex, and ten times with the female as ever, studies have not explicitly demonstrated a deleterious the donor sex. Experiments where no mating pairs formed effect on their host. In experiments where realistic numbers were repeated. of mites have been placed on hosts under controlled conditions, no cost to the host has been demonstrated (Cantwell et al., 1985; Hochmuth et al., 1987), and obser- Results vations suggest that, in other cases, the effect of mites on host fitness may be minor (e.g. Husband & Sinha, 1970). More mites transferred to individuals that had mated with We here investigate the biology of the podapolipid mite an infected partner than to those that did not (Table 1). Coccipolipus hippodamiae McDaniel & Morill on the The null hypothesis that individuals that had mated with coccinellid beetle . Coccipolipus hippo- an infected partner received the same number of mites as damiae is an ectoparasite of adult ladybirds, engorged individuals mated with an uninfected partner was rejected females being found on the underside of the elytra, towards (Wilcoxon signed rank tests: for male to female transfer, the anterior, with larval females being found on the n = 6, T= 0, P < 0.05; for female to male transfer, n = 0; underside of the elytra, towards the posterior tip. We T = 0; P < 0.005). Additionally, the null hypothesis that show that this organism is a sexually transmitted parasite individuals that had mated with an infected partner received of the coccinellid beetle A.bipunctata and discuss the the same number of mites as individuals which did not significance of this finding. mate was rejected (Wilcoxon signed rank tests: for male to female transfer, n =8, T= 2, P

Table 1. The number of mites transferred to thc individuals of the recipient sex, either mating with partners of different status with respect to infection, or failing to mate, in the experiment described in the text. Dashes indicate no interaction occurred.

(i) Transmission to males in the presence of two infected and two uninfected females

Mean no. of mites Mean no. of mites Mean no. of mites transferred to males transferred to males transferred to males which mated with an which mated with an which did not mate Experiment infected female uninfeeted female during the experiment

C 13 0 0 D 5.5 0 0 G 68 0 0 H 14 1 2 J 18 0.5 - K - - 0 0 13 0 0 P 29 0 0 Q 42 1 0 T 6.5 0.5 - U 81 1.5 0

(ii) Transmission to females in the presence of two infected and two uninfected males

Mean no. of mites Mean no. of mites Mean no. of mites transferred to females transferred to females transferred to females which mated with an which mated with an which did not mate Expcriment infected male uninfeeted male during the experiment

A - 0 0 B 17 0 0 E 7 - 1.5 F 4 0 0 I 23 1 1.5 L 6.5 1 0 M 13 0 18 N 16.5 - 0 R 16.5 0 - S 12 - 1

pair which was to serve as the uninfected control was each day was recorded, and clutches were allowed simultaneously subjected to exactly the same conditions to hatch naturally. The hatch-rate of these eggs was and manipulation as the infected individual, save for the recorded. Females were remated every seventh day to physical transfer of mites. Each individual was then placed prevent infertility arising from sperm depletion (Majerus, in a separate petri dish, to prevent any later transfer of 1994a), members of each pair again being crossed to mites. unrelated, uninfected, sibling males. The dishes were The females were thenceforth maintained on a feeding placed individually on the bench to prevent the differences regime of seven large A.pisum every 2 days, alternating in fecundity that may arise from to the effects of light when daily provision between four and three aphids. This level dishes are stacked. of aphids provides enough for one small clutch of eggs to The effect of mites on fecundity and egg fertility was be laid daily. The mites were given 7 days to establish measured over a 25-day period. At the end of this period, on the female ladybirds, and then both members of the the number of mites on each individual was scored. There- pair were mated. Males used in matings to a given pair after, each member of the pair was fed excess aphids for were brothers, unrelated to the females, and free from 7 days, and the viability of eggs recorded. After this time, mite infection. Following mating, all ladybirds were the individuals were placed back upon the alternating provided with a clean petri-dish every day to control for three then four per day diet until death, and the the stimulation to oviposition produced by changing viability of eggs recorded. The longevity of individuals was dishes (Majerus et al., 1989). The number of eggs laid on recorded. Sexually transmitted disease iti A. bipirrictuta 233

Table 2. The effect of mite infection on the rate of egg laying, the fertility of eggs laid, and the longevity of female A. bipunctuta

Infected with mites Not infected with mites

Lifcspan of Lifcspan of Eggs laid Proportion of adult female Eggs laid Proportion of adult female Pair in 25 days eggs hatching (days) in 25 days cggs hatching (days)

2 213 0.52 74 364 0.93 68 3 294 0.20 92 285 0.76 68 4 21 1 0.24 85 285 0.95 119 5 136 0.29 92 345 0.68 66 6 245 0.08 142 233 0.86 83 7 15 1 0.44 60 293 0.56 103 8 141 0.45 74 267 0.84 118 9 282 0.14 77 254 0.81 90 10 277 0.00 77 278 0.89 123

Results when first laid, but shrivelled and became translucent within 24 h. Normal eggs remain opaque and maintain The fecundity of infected individuals was significantly their shape until hatching. The inviability of eggs from lower than that of their controls (Table 2) (paired t-test: infected individuals was maintained even during the t= 2.52; 8d.f.; P0.5). The effect of mites on the fertility of females The viability of eggs laid by females infected with the from the Moscow population mite decreased markedly with time. At first, eggs laid by Method infected individuals had a good level of viability, but this declined, frequently to zero, within 7-9 days of the start Seven laboratory reared adult female ladybirds derived of the experiment (Fig. 1). The eggs laid by mite-infected from the Moscow population were each infected with six ladybirds after this period were a normal opaque yellow larval mites as described previously, and the females fed

A. Mite infected individuals B. Control individuals

1-5 6-10 11-15 16-20 21-25 1-5 6-10 11-15 16-20 21-25 Time period following first Time period following first copulation (days) copulation (days)

Fig. 1. Hatch-rate of eggs laid by fcmalc A.bipunctutu bearing mites (A) and individuals free from infection (B) during sequential 5-day periods. 234 Gregory D. D. Hurst et al. on the same alternating daily four aphid-three aphid probably different from those observed on adults in the ration. After 7 days the females were mated, and the field, because the ladybirds were confined in single-sex fertility of the egg clutches produced monitored in compari- dishes between experiments. If transfer of mite larvae son to the fertility of eggs from uninfected siblings on a takes place mainly during copulation, maintenance in similar regime. Each female was re-mated every 7 days to single-sex dishes might produce unusually high mite an uninfected male. densities upon the host. Furthermore, as the number of larval mites builds up on unmated adults, mites are seen to crawl over the surface of the ladybird and into the petri Results dish (Hurst, pers. obsn). Therefore, using rarely mated adults might increase mite transfer rates to non-sexual- The mites had a similar effect on the fertility of females partner ladybirds. However, although the experiments from the Moscow population as those from the Cambridge may have overestimated the amount of transmission population. Whereas the control individuals showed high between non-mating motile ladybirds, as found in spring egg hatch-rates throughout their lifespan, with at least or summer, the mite may transmit non-sexually during 50% of the eggs hatching in every clutch, after on average periods of diapause or aestivation, when they often aggre- 9.5 days (SE 1.0), all the eggs in clutches laid by the seven gate (Majerus, 1991, 1994b). As yet it is impossible to say infected females shrivelled rapidly and failed to hatch, whether sexual transmission is sufficient in itself to maintain although yellow and intact when first laid. the infection in the population, or whether horizontal transmission unassociated with sex is also necessary and important. Discussion The above study also provokes questions as to the cause of female sterility. It is hard to believe that the eeg The mite Coccipolipus hippodamiae is transmitted between inviability is a direct effect of the energy sap that the A. hipunctara partners during copulation. Tests show this mite represents, given the modest decrease in the number infection to have a detrimental effect upon the lifetime of eggs laid. There are perhaps two possibilities. First, that reproductive success of adult females which bear it. The the mite either uses or interferes with the production of a mite appears to produce roughly a 25% decrease in the particular nutrient required for host egg fertility. The number of eggs laid by infected ladybirds. In addition, the collapse of eggs laid by mite-infected individuals is perhaps mite produced a massive decrease in the viability of the symptomatic of an ineffective chorion; removal of this eggs laid by their host. The finding of a similar reduction in layer is known to produce egg death by drying (Ricci & fertility and egg viability for infected females from the Stella, 1988). Alternatively, the effect may not be due to Moscow population showed that this effect is not a result the mite directly, but due to pathogenic micro-organisms of introduction of the mite to a previously uninfected host which it introduces to its host. Insect resistance to infect- population, but a real effect of the mite on A.bipunctata in ion comes largely from the gut and cuticle barrier, and general. Therefore A.bipunclata may be considered to parasitoid wasps have been recorded as vectors of a diverse have a sexually transmitted disease of unusual severity. array of pathogenic micro-organisms, from microsporidia The effect of the mite on males is more difficult to of the genus Nosema (Siege1 el al., 1986). to viruses ascertain. The most probable effect would be a general (Irabagon 8c Brooks, 1974; Beegle & Oatman, 1975) and reduction in vitality and vigour which would reduce the bacteria (Bucher, 1963). Ectoparasitic mites may cause ability of males to locate and attract partners, but this similar disease transfer, and represent an important source would be difficult to quantify under the artificial conditions of new infections. of the laboratory. However, even if the mite does not have Perhaps the most important sphere in which this study any direct negative fitness effect on male hosts, it will has importance is in discussions of the population biology still reduce their reproductive success, as it would be of infectious diseases in invertebrates. This disease affects transmitted to their partners, and thus reduce their fertility. only adults, and sexual transmission appears to be of key Our experiments show that A.bipuncrata from Moscow importance in its maintenance (but see caution above). has a sexually transmitted mite which has negative fitness The case is similar to the peculiar case of the sexually effects on its host. Is mite transmission confined to periods transmitted ectoparasitic nematode of noctuid moths. of mating? The experiments above revealed a low level of Such diseases must be expected to be rare in invertebrates, transmission to non-sexual-partner hosts (Table 1). The for as well as requiring host promiscuity (which is certainly question arises as to whether this is an artefact of laboratory true of A.bipunctata: Majerus, 1994a), they also require conditions. There are two reasons to think it is. First, either significant overlap of generations, with inter- ladybird density is much lower in the field than in the generational matings, or an alternate host to exist. Without confined experimental boxes. Observations of the mite an alternate host, years in which no overlap of generations suggest it has limited longevity away from the host: mites occurs would cause local extinction of the sexually trans- placed in clean petri dishes in the laboratory live less mitted organism. This is perhaps where the podapolipid than 12 h (Sharpe & Hurst, pers. obsn). Therefore mites mites represent a unique case. Whereas Noctuidonema, leaving the host may have a very low probability of finding the sexually transmitted nematode, is found on many a new host. Second, the density and behaviour of larval species of noctuid moths (Remillet & Silvain, 1988; mites on the hosts in the sexual transfer experiments are Rogers et al., 1990a, b), Coccipolipus species are known to Sexually trarismitted disease iri A.hiprrrictata 235

be very limited in their host range within coccinellid due to the death of the mite colony (Hurst, pers. obsn). beetles (e.g. Schroder, 1979). Whereas in Noctuidorzemu, This might allow the transmission of any novel bacterial other species may act as a pool for re-infection following infection to progeny. Clearly, work on the role of mites local extinction, the paucity of alternate hosts for members such as the podapolipids in vectoring inherited parasites of the genus Coccipolipus suggests a greater reliance on (both bacteria and transposable elements) would be overlap of host generations. The importance of overlap of timely, in this and other systems. generations is also consistent with the limited observations of Chippodamiae. It is known to be present in Moscow, but not Cambridge. In Britain, A.bipunctata has one main Acknowledgments generation per year, with a partial second generation in many years produced partly by inter-generational pairings We thank Dr Sally Corbet, Dr Bryan Grenfell and Dr (Majerus, 1994b). However, from time to time inter- Charles Godfray for helpful advice, and Mr Roger Day generational crosses are prohibited because the over- and Mr Alan Feast for technical assistance. The work was wintering generation dies out before their progeny reach carried out in a laboratory funded by the Wolfson Found- reproductive maturity. In Moscow, however, three gene- ation, and funded by a Junior Research Followship at rations of A.bipunctutu may be observed in a year Christ’s College, Cambridge (G.H.), an N.E.R.C. grant (Zakharov, pers. obsn). (GR9/993) (M.M.) and a grant from the Russian Foun- The finding of a sexually transmitted organism also dation of Basic Fundamental Research (I.Z.), for which merits consideration in an evolutionary light. The we are grateful. presence of an STD may be expected to have an evolut- ionary effect on host sexual behaviour (Borgia & Collis, 1989; Hamilton, 1990; Sheldon, 1993). The study of mate References choice in A.bipunctata has a long history (see Ritchie, 1992, for review). The presence of an STD adds an extra Anderson, R.M. & May, R.M. (1991) Infectious Diseuses of dimension to this study. Females which can avoid mating Humans: Dynamics and Control. Oxford University Press. with infected males will clearly be favoured by natural Beegle, C.C. & Oatman, E.R. (197.5) Effect of a nuclear poly- selection, as may male selection of uninfected females. An hcdrosis virus on the rclationship bctwecn Trichoplusiu ni elaborate mate rejection behaviour is observed in A. (Lepidoptcra: Noctuidac) and the parasitc. H.vposrer exiguue (Hymcnoptcra: Ichneumonidac). Journal of Invertebrate bipunctata: a female may prevent intromission by pulling Pathology, 25, 59-71. her abdomen up under her elytra, and may use various Borgia, G. & Collis, K. (1989) Female choice for parasite-free physical means (rolling over, kicking, raising legs, running male satin bowerbirds and the evolution of bright male plumage. (Majerus, 1994b)) to displace an unwanted suitor. Obser- Behavioral Ecology and Sociobiology, 25. 44-454. vations of initial stages of interaction between male and Bucher, G.E. (1963) Transmission of a bacterial pathogen by female, and later possible rejection behaviour, suggest the ovipositor of a hymcnoptcrous parasite. Journal of lnsecr that the female may be testing the male’s vigour (see Pathology, 5, 277-283. Majerus, 1YY4b, for review). If this is true, then it may be Cantwell, G.E.. Cantclo, W.W. & Cantwell. M.A. (1985) Effect that the interaction between male and female is the evolut- of a parasitic mite, Coccipolipus epilachnae, on fccundity, food ionary product, in part, of the need to avoid disease. Mate consumption and longevity of the Mexican bean bcctlc. Journal of Entomological Science, 20, 199-203. choice may, in part, derive from the need to avoid infection Gauchat, C.A. (1972) A notc on Podupolipoides grassi Bcrlcsc with parasites (Borgia & Collis, 1989). Clearly, this awaits (Acarina: Podapolipidac), a parasite of Cliortoiceres terminferu test. Walker. thc Australian plague locust. Journal oftlie Austruliun The evolutionary effect of this mite on its host may go Entomological Society, 11. 259. beyond the design of mating behaviour. Mites have been Grimstad, P.R., Ross. Q.E. & Craig. G.B.. Jr (1980) Aedes considered a potential source of novel infections with triseriatus (Diptera: Culicidae) and La Crossc virus. 11. Modifi- transposable elements and inherited symbionts, these cation of mosquito feeding behaviour by virus infection. Journal having only a short life outside the host (Houck et al., of Medical Enromology, 17, 1-7. 1991). Adalia bipunctata is host to inherited parasites, a Hamilton, W.D. (1990) Mate choice ncar or far’? American bacterium of the genus (Hurst eta/., 1992, 1993; Zoologist, 30. 341 -3.52. Hochmuth. R.C.. Hellman, J.L.. Divcly, G. & Schrodcr. R.F.W. Werren et ul., 1994), and one of the genus Spiroplusma ( 1987) Effect of cctoparasitic mite Coccipolipus epiluchnue (Hurst et a/., 1995). It is notable that members of the Acari (Acari: Podapolipidac) on feeding, fccundity and longevity of do bear a similar to that which causes male- soy-bcan adult Mexican bean bcctlcs (Coloptcra: Coccincllidac) killing in Moscow A.bipunctuta (Tully et al., 1981; at diffcrcnt tcmpcraturcs. Journal of Economic Entornology. Weisburg et al., 1989). 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