Investigations on the life cycle and morphology of Tunga penetrans in Brazil
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- Nagy, N., Abari, E., D’Haese, J. et al. Parasitol Res (2007) 101: 233. doi:10.1007/s00436-007-0683-8
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In the present study, the life cycle of Tunga penetrans was established in Wistar rats in the laboratory, and the morphology of the resulting developmental stages was studied by means of light and scanning electron microscopy. It was seen that the females enter at a nonfertilized stage through the skin of their hosts. Only there the copulation occurs, while females and males brought together in a Petri dish showed no interest in each other. In any way—fertilized or not—the females start about 6 days after penetration and hypertrophy with the ejection of eggs. While fertilized eggs proceed to development, the unfertilized ones remain arrested. The eggs are ovoid and measure about 600 × 320 μm. The larvae hatch from the eggs 1–6 days (mean 3–4) after ejection. Formation of larvae 2 took at least another day, while 4 up to 10 days more were needed until this larva starts pupation (mean 5–7 days). The formation of the adult fleas inside the puparium occurred within 9–15 days (with a maximum hatch at day 12). Adult female fleas having reached the skin of a host start blood sucking within 5 min and prepare to enter the skin. After 24 h, the flea stacked already with two thirds of its body inside the skin. After 40 h, the penetration was completed, and feeding and hypertrophical enlargement started, which was completed on day 6, when eggs became ejected. When studying the morphology of the fleas obtained from different hosts, slight variations were seen, which, however, are not significant for a species separation but may be an indication of the presence of different strains/races or the beginning of such a formation.
Worldwide, about 3,000 flea species are known (Lewis 1998). All have piercing and sucking mouthparts by which they suck blood of their hosts. The host spectrum includes about 94% mammals and 6% birds. Normally, 99% of the flea population live (as larvae and pupae) on the ground and were not perceived by humans. Thus, host attacks are only done by 1% of the living flea population (Linardi and Guimarães 2000).
Some flea species can transmit human pathogens; however, the complete spectrum of microorganisms is not known. Recently, it was found that C. felis can transmit viruses such as the feline leukemia virus or Calici virus (Vobis et al. 2003, 2007). The aetiology agent of plague, Yersinia pestis, is transmitted by the rat flea Xenopsylla cheopis but also by other species (Achtman et al. 2004). Other flea species transmit in addition bacteria (Feldmeier et al. 2002; Titball et al. 2003), tapeworms (Mehlhorn et al. 1995), filarial nematodes (Linardi and Guimarães 2000), or a variety of Rickettsiae (Blair et al. 2004).
Sand fleas of the genus Tunga initiate inflammatory reactions after penetration in the epidermis of the hosts. In the human host, Tunga penetrans lesions are frequently superinfected by aerobe and anaerobe bacteria (Feldmeier et al. 2002; Greco et al. 2001).
With a length of 1 mm, the sand flea, T. penetrans, is the smallest flea species known (Geigy and Herbig 1949; Connor 1976). In contrast to most of other flea species, the genus Tunga occurs only in the tropics (Heukelbach et al. 2001). Tunga species (especially T. penetrans) have a wide host range including cats, dogs, pigs, donkeys, monkeys, and rodents (Heukelbach et al. 2004). Two species parasitize humans namely, T. penetrans and the recently discovered species Tunga trimamillata (Hicks 1930; Eisele et al. 2003; Muehlen et al. 2003; Pampiglione et al. 2002, 2003, 2004).
Flea species have particular morphological characteristics and can easily be identified by means of a stereomi-croscope (Lane and Crosskey 1993). However, this is not easy for the genus Tunga where it is difficult to differen-tiate species on the ground of morphological criteria (Pampiglione et al. 2004). Deoxyribonucleic acid (DNA) analysis of regions coding several parameters have shown that at least in Northeast Brazil, T. penetrans is probably a complex with closely related subspecies (Vobis et al. 2004; Nagy et al. 2007).
Sand fleas have, in contrast to other flea specimens, well-developed laciniae and an epipharynx, which are important for females with respect to their invasive way of living. The head is flattened and has no ctenidial combs, which are found in some other species like C. canis or C. felis.
Materials and methods
Study area and sample collection
The study was conducted in Fortaleza, the capital of Ceará State, Northeast Brazil. Adult T. penetrans were collected from the soil and from skin of hosts, in the shanty town Morro de Sandra’s and the village Barra Velha, endemic areas for tungiasis. T. penetrans larvae were sieved from the sand in Morro de Sandra`s and later after having established the life cycle experimentally, also in the laboratory. Adult stages of both sexes were collected from different hosts. Hypertrophied female T. penetrans were removed surgically from the epidermis of different hosts. Collection of specimens in the environment took place from September to December 2004, a period of the year when the transmission has its peaks (Eisele et al. 2003).
Sand samples of 100 to 200 g each were taken from three different ground depths (1, 5, 10 cm) at different locations inside and outside the houses of Morro de Sandra’s. A form was filled out for every sample in which soil and air temperature, humidity, hygiene level of the houses, and other environmental variables were recorded. Ground temperature and moisture were measured by a hygro-thermometer (HT 100 Voltcraft, ORT Germany). Air temperature and humidity were measured by an infrared thermometer (H1 Testo, ORTGermany).
Eighty laboratory-raised adult female Wistar rats (∼200 g) were placed in cages in Morro de Sandra’s at locations where tungiasis was common in humans. Animals were examined every day. If three or more embedded sand fleas were present, the animal was transferred immediately to the laboratory. All laboratory experiments were realized at the Universidade Federal do Ceará in Fortaleza.
Measurements of the different Tunga stages were done with a Hi-Scope Advanced KH 3000 digital microscope (Hirox, Tokyo, Japan).
Life cycle in the laboratory
Expelled eggs were collected by positioning infested Wistar rats on black paperboard. Eggs were counted and kept under various environmental conditions in Petri dishes. When larvae hatched, they were also maintained under different environmental conditions. The cocoons were separated from the sand, and their development was followed by microscopy in a separate Petri dish. The time span between excretion of eggs and the appearance of adult fleas was recorded.
Experimental infection in the laboratory
Laboratory-raised adult male and female sand fleas were kept for 5 min at −10°C and then placed on the feet or the tail of a rat by use of a fine painting brush. Duration of penetration into the epidermis were documented.
Stages of development after penetration were classified according to Eisele et al. (2003) using the Fortaleza classification.
Copulation of the fleas
Laboratory-bred females T. penetrans, which never had come into contact with a male sand flea, were placed on rats to see if unfertilized females borrow into the skin. To examine, whether free-living stages of Tunga copulate or not, separately raised male and female sand fleas were brought together in Petri dishes and the way and frequency of contacts between the fleas were documented.
Morphological examinations were carried by means of light and scanning electron microscopes of all T. penetrans stages. Embedded females were examined in different stages after penetration.
For scanning electron microscopy specimens were fixed in 5% glutaraldehyde, washed with 0.1 M cacodylate-buffer and afterward dehydrated in an ascending concentration of acetone (from 20 to 100%). The dehydrated specimens were dried by a critical-point dryer (Balzers Union, ORT Germany). To raise surface conductance, dried fleas were gold sputtered at a pressure of 0.1 mbar and a voltage of 25 mA for 3 until 5 min with a cathode-sputter (Balzers Union, ORT Germany). The objects were examined with an AMR 1000 scanning electron microscope (Leitz, Wetzlar, Germany) and photographed with a Leica MD-2 (Leitz, Wetzlar, Germany).
Investigations on the life cycle
Seventy-five people, who live in the favela Morro de Sandra’s were examined for penetrated Tunga. More than 55% of these people were afflicted and showed Tunga-caused lesions at their skin. The afflicted persons were mostly children in the age from 2 to 8 years. Most highly afflicted adults were young women, who spent the whole day at home. Humans and groups of five different host animals (dogs, cats, pigs, rats, and mice) of Tunga were examined to specify the location of the penetrated female sand fleas. A total number of 236 invaded Tunga was investigated, 84.3% of which were found in the feet of the hosts. Only 15.7% of the sand fleas had penetrated elsewhere preferring the mouth or the abdomen of their hosts, and in humans, 14.5% had penetrated into the hand. This is the background from where adult sand fleas and larvae were obtained for the laboratory experiments.
Occurrence of Tunga larvae
In the previous named locations, 131 sand samples inside and outside the dwellings were taken in different depths, from the surface down to 10 cm. In the samples, 88 Tunga larvae were found. Fifty-four Tunga were found inside the houses, and 34 were found in the sand outside the houses. It was noted that 58% of the larvae were found in the layer between 2 and 5 cm below the sand surface. The other larvae (except for three) were found in the layer of the surface down to 2 cm. Outside the dwellings, the larvae were hidden in deeper sand layers than inside. Most of the Tunga larvae inside the buildings were found near to the sleeping facilities and cooking places. The larvae outside the dwellings were found at the normally sleeping places of the animals and at those places, where the children play.
Size measurement of different Tunga stages
It was shown, that females were smaller with respect to nearly all measured criteria, except for the epipharynx and the maxillar palp.
Life cycle in the laboratory
Wistar rats exposed in Morro de Sandra’s had a median of five embedded sand fleas (range 4–7). A total of about 1,000 of eggs were expelled by the four to seven embedded sand fleas per day. Female sand fleas in stage II to III expelled on the average 15 eggs per hour, which were being transferred into different growth media. The eggs were incubated separately within a period of 2 until 10 min or after 3 min after egg ejection (about 500 eggs were used for each of the tests in different media/vessels: empty Petri dishes, Petri dishes filled with sand from the favela, Petri dishes with boiled sand from the favela, and Petri dishes filled with wet cotton wool and paper board). On the average, 15% of eggs developed into larvae. Eggs bred in empty Petri dishes became dry very fast and showed no further development. The eggs in the favela sand also did not develop. The most effective method for the development of the sand flea eggs was to breed them on permanent wet paper board. Female sand fleas collected in the favela and placed on rats in the laboratory did only expel nonfertilized eggs. Thus, egg production is not a reaction of fertilization.
The time needed for the larval development inside the egg was 1 to 6 days after egg expulsion; most hatched on days 3 and 4. After day 6, no more larvae hatched. The transformation of L1 to L2 needed just a few hours (median 10, range 8–24 h).
The hatched larvae were studied under different conditions to clarify further development. Nutrient solution for flea breeding (Bayer Leverkusen, Germany) was used. A nutrient solution was mixed with minced adult Tunga, with boiled or not boiled favela sand, or with sand without the nutrient solution and no minced adults. A total of 1,691 larvae was used in the different tests. Only 14.3% initiated a further development and formed a pupal cocoon. Cocoon formation started 5 to 11 days (median 7 days) after hatching from the egg. Out of 118 cocoons, 53 adult sand fleas hatched. The nutrient solution with minced adults and boiled favela sand was the most effective environment. In Petri dishes without sand, no larvae built a cocoon. Petri dishes filled only with favela sand showed a 2-day retardation in pupal development.
The development inside the puparium to adults took between 9 and 15 days (median 12 days).
Infestation in favela
For the infection study, at the beginning, 80 rats had been exposed in cages in the favela of Morro de Sandra’s. Of them, 53 rats were analyzed for on-host development of the penetrated fleas. From the day after exposition, the rats became infested by the female Tunga; most of the rats were afflicted with more than five female sand fleas on day 6 after they had been brought to the favela. The majority of rats became soon infested with more than 25 female sand fleas. However, a few rats being exposed for more than 10 days showed only three Tunga.
The infested rats were controlled every second day with respect to the penetration stage referring to the Fortaleza classification (Eisele et al. 2003) and confirmed these findings from the field.
To monitor all stages of Tunga penetration and hypertrophy in accordance to the Fortaleza classification, the female Tunga were placed in the laboratory onto narcotized rats.
At the maximum 5 min after taking the sand fleas onto the rats, they began to suck blood and penetrated into the skin. They sank their head toward the skin of the rat, whereas their abdomen made an angle of 45° to the skin of the host. The head of the flea was totally embedded into the epidermis after 1 up to 8 h. These Tunga stages appeared darker, and the zone between abdominal segments 2 and 3 was stretched. After 24 h, the flea was embedded with two thirds of its body into the epidermis of the rat, while the females had nearly completely sunken into the epidermis after 40 h with only the two last segments remaining above the skin surface showing the genital pore and the stigmata.
Three days after the penetration of the females, the skin around the penetration site became concave and first signs of inflammation were seen. The hypertrophy of the sand flea increased beginning just after first step of entering. On the fourth day after penetration, the intersegmental membrane between abdominal segments 3 and 4 was considerably enlarged in size resulting in the impression of a life belt. A distinct swelling of the flea body and the beginning of feces production was seen from day 5 after penetration. On day 6, the hypertrophy had reached its peak, and the expulsion of eggs started. A pronounced inflammation around the penetrated flea was clearly visible. The hypertrophied abdomen disrupted beginning 8 to 9 days after penetration. The secretion of eggs and feces stopped finally in all specimens on day 25 after penetration. One month after penetration, the female died and was removed within 2 weeks by skin repair mechanisms. A circular deepening remained visible for a month.
Five Wistar rats were infested with 25 adult female fleas raised in the laboratory. Rats were anesthetized as soon as the sand fleas were completely embedded in the skin. Then, five males were placed onto each rat close to the places where females had penetrated. Male Tunga rapidly (within seconds) found the penetrated females. At first, they ran around the female, and then they placed their copulation organ in direction to the upright abdominal end of the female. Thereafter the copulation began. The copulation took 2 min at the maximum, mostly only a few seconds. It was repeated two to three times with the same female, then the male Tunga changed to another penetrated female for a new copulation. After the copulation, the male Tunga sucked blood from the rat for several seconds, which was proven by its occurrence in the intestine.
Investigations of the morphology
Life cycle stages of Tunga
The head of the larva was provided with a so-called eggshell breaker, which enables the larvae to disrupt the eggshell. After molt, the eggshell breaker disappeared. The antennae of the larvae were bar like and stretched away from the head. First rudiments of the maxillary palp and the labrum could be seen. Eyes were not present during this phase of development. The last abdominal segment was divided into two lobes, which both carried an anal brace.
Comparison of adults obtained Tunga from different host animals
Ten males and females from each host group (humans, dogs, cats, rats) were examined for morphological differences. The shape of the head and abdomen was studied because differences were expected to occur mainly there.
Adults collected from pigs showed in all cases the second type of head morphology (Fig. 13). In contrast, sand fleas obtained from dogs had a more or less straight front of the head (Fig. 14). The head front of Tunga collected from cats was very similar, while adults collected from rats showed a very linear shape of the head. Only at the top of the nose a little bulge became visible.
It was not possible to find differences along the abdomen of the different fleas from different host, as they were embedded in the skin. Furthermore, with increasing hypertrophy, the shape of the abdominal end of the females changed. Thus, there is no statement possible on the differences of the abdomen of the fleas of different hosts. The male Tunga did not differ much in their abdominal shape, so that there is also a clear statement when comparing the Tunga males from different host animals.
The genus Tunga belongs to the family Tungidae, which comprises ten species (Pampiglione et al. 2003). Rodents were used as hosts by six Tunga species, and armadillos were hosts of two Tunga species (Pampiglione et al. 2002). Some species are very host specific like Tunga bondari, which afflicts only the anteater Tamandua tetradactyla. The species T. penetrans in contrast has a very wide host spectrum; it includes rodents, cats, dogs, sheep, monkeys, donkeys, pigs, elephants, and humans (Heukelbach et al. 2004; Njeumi et al. 2002).
The females of the genus Tunga are known along the tropical and subtropical belt as a very painful nuisance because of their invasive way of living. The fleas from six examined host species in this study showed frequently the same shape of penetrated Tunga females. It was assumed that the visible cluster was produced by the penetrated Tunga (Eisele et al. 2003). Studies made on the life cycle of Tunga in 2004 lead to another explanation for this cluster formation. Sand fleas cause inflammation starting a few days after penetration into the skin of the host, then the tissues excrete water as a reaction at the inflammation, and thus the skin is softer at this side. Because Tunga prefers to penetrate at the soft locations, the cluster formation might be explained.
The paws showed the highest number of fleas because of their close contact to the ground. Older domestic animals of cats and dogs had less penetrated sand fleas than the youngsters because of the thicker skin at their paws.
Rats exposed in the favela showed a high infestation rate with Tunga within a week. This indicates a high incidence of free-living female fleas, which was confirmed by the permanently afflicted persons living in these rural suburbs (Heukelbach et al. 2004; Feldmeier et al. 2003; Muehlen et al. 2003).
The residents of the shacks, who had water supply, sanitary equipment, and a concreted ground, had less problems with the Tunga nuisance than others who had a lower hygiene level. The results concerning the examined sand from the favela support the statement that hygiene is an important factor for sand flea prevention. In shacks with concreted grounds being cleaned every day with water, Tunga larvae were hardly found. In shacks with a sandy ground, the number of larvae increased. The whereabouts of the Tunga larvae are in direct correlation to the adult fleas, which were mostly found near the sleeping facilities and cooking places or near the rubbish. The number of persons living in a shack also plays an important role for the rate of Tunga infestation. If people are living close together, they are afflicted to a higher degree than others living in a larger distance. Families with pets were also more afflicted by sand fleas than families without pets.
Tunga larvae were found in the sand in a depth of 2–5 cm. This was expected because the sand surface was too hot and too dry for larval development, while deeper layers of the sand offered not enough oxygen supply or had a soil humidity of 80%. Freshly hatched larvae had a very thin skin and would quickly drain on the sand surface in direct sun. Because they have to visit the surface to feed, larvae were found only at shady places. Difference in temperature was only 0.7°C between inside and outside the shacks. Thus, this was not a determining factor for the places where larvae live. Further studies of larval behavior must be done including also their vertical migration in the sand.
Larval measurements showed a correlated growth of the larvae, except for the larvae on the second day that were in mean 350 μm smaller than the larvae measured in the first day after hatching. The development from larva 1 to larva 2 was accomplished within less than 24 h. This supports the assumption that the size differences between days 1 and 2 are due to the transformation of the larvae.
Size measurements of adult Tunga showed that females are smaller than males in all extremities except for the epipharynx. The maxillary palp of females was only a little bit smaller than that of the males. The nonproportional size of the mouthparts of the female fleas might be explained by their invasive way of living.
In the laboratory, several strategies had been chosen to produce the sand flea eggs. The way of Hicks (1930), who used just cooked street dust, did not lead to formation of larvae. The most effective method in our experiments to produce larvae was the storage of eggs on permanently moist paper board. This is in contrast to the finding of Hicks (1930).
The percentage number of fleas, which develop from egg to an adult flea, was very low. Only 15% of the eggs developed to larvae, and from these larvae, only 14% finally formed a cocoon. Only half of the flea’s pupae gave rise to adult fleas in a sex ratio of 40% males and 60% females.
The question is not solved whether the small number of fleas achieved from laboratory breeding is due to the breeding conditions in laboratory or whether the output under normal conditions is as small or even smaller.
In the present laboratory breeding of the Tunga fleas, it was found in contrast to the literature (Geigy and Suter 1958) that also nonfertilized eggs were ejected by the females. Until now, it was believed that only fertilized eggs were excreted by hypertrophied Tunga females. Furthermore, in the past, it was suggested that only fertilized Tunga female penetrate into the skin of the host. The present copulation study proved that free-roaming adult Tunga of both sexes did not copulate. Thus, the statements of Geigy and Suter (1958) were supported by our observations. Geigy and Suter (1958) observed freshly hatched adults for a week below a glass and could not observe any copulation. This is in accordance to our results. Geigy and Suter reported in addition that the act of copulation takes about 20 min and that the male Tunga takes a blood meal during the copulation on the host. In our study, it was noted that the copulation takes about 2 min at the maximum. In general, the copulation was done in a few seconds, and a blood meal was taken by the male just after copulation.
In the present study, similar steps of penetration of the Tunga were seen as described in the Fortaleza classification (Eisele et al. 2003). The Fortaleza classification defines that phase 1 of the penetration lasts 3–7 h. In our observations, a complete penetration into the skin occurred but, in some laboratory cases, only within 40 h.
Studies on molecular differentiation of single T. penetrans (Vobis 2002; Vobis et al. 2004, 2005) revealed differences. These studies showed differences in sequence between the different host animals from T. penetrans of about 49% when probing the mt16S ribosomal DNA. This may suggest that there might exist several species or races of T. penetrans.
It was proposed that such a big difference in sequence must also have a morphological correspondence. Therefore, further investigations on morphology were done during the examinations in the present study.
Morphological comparison of stages of T. penetrans stages from five different hosts were proceeded by means of scanning electron microscopy with comparing the front of the head and the posterior end of the flea.
The fleas, which were collected in 2002 from humans, showed remarkable differences in the shape of the head (Vobis et al. 2005), which were also seen in a few T. penetrans of the present study. A ski-jump ramp-like-shaped front of the head was documented in one group, and a more rounded head front was observed in the other. Sand fleas from pigs showed a tapered end of the head with a distinct ski-jump-like shape. Fleas from rats, cats, and dogs did not show such characteristics in the shape of their heads. They all had a little sloped shape of the head with a more or less straight appearance of a nose.
Some penetrated Tunga showed a deformed head because it was overlapped by their hypertrophied abdomen. Because of chitin reinforcements, the typical cloverleaf structure of hypertrophied T. penetrans was seen (Geigy and Herbig 1949).
Differences by means of the shape of the abdomen could be seen neither in male nor in female Tunga from different hosts. However, it is not clear whether these slight morphological variations are expression of an emerging subspecies.
We are grateful to Prof. Trentini and Prof. Luchetti from the Facoltà Di Medicina Veterinaria in Bologna (Italy) for sending us three specimens of gravid female T. penetrans and three specimens of gravid female T. trimamillata isolated in Ecuador. Furthermore, we thank Valeria Santos (Fortaleza) for her support during the field study in Brazil.