Very few studies regard the biology of the larva and its effect on the woodlouse host. These studies usually demand a long period of time due to the difficulty of obtaining the parasitoids (Thompson 1934, Bedding 1965, 1973). This difficulty is partially explained by the low prevalence of this parasitoid on natural populations and for the apparent specificity of host species (Bedding 1965). Prevalence on natural populations is usually lower than 2% and seems to be associated with the infection method.

Adult Rhinophoridae flies copulate and the female deposits the eggs on substrates (Bedding 1965, Wijnoven 2001) contaminated by uropod gland secretion of isopods rather than on the host itself (Bedding 1965) which may be a derived character in this group of parasitoids (Wood 1987). This secretion is not commonly observed in all woodlice species but it is rather easily obtained from Porcellio scaber Latreille, 1804 (Gorvett 1946, Deslippe et al. 1996) which might explain why this species has the highest number of known parasitoids and highest prevalence on natural populations (Bedding 1965, Sassaman and Pratt 1992).

The eggs deposited on the soil hatch and the 1^st instar larva attaches itself to the body of a passing woodlouse. The larva may wave its anterior end slowly forward and sideward in an attempt to attach itself to the body of a passing woodlouse (Pape and Arnaud 2001). This method of infection is affected by host size since the larva cannot reach the sternites of bigger (taller) animals. It was also observed the suitability of the host relates to a specific period of the molting cycle of the isopod. Differently from insects, crustaceans present a highly calcified cuticle (Roer et al. 2015). Within crustaceans, isopods have developed specific strategies to recycle calcium from the old cuticle such as a biphasic molting (they first molt the posterior half and then the anterior half of the body) and accumulation of amorphous calcium carbonate in the anterior sternites prior ecdysis (Greenaway 1985, Steel 1993, Ziegler 1994). The fly larva attaches itself to isopods with calcium plates (i.e., during premolt or intramolt) and penetrates through the intersegmental membrane of the sternites of the freshly molted host (Bedding 1965), since they present a softer cuticle at this stage. Nonetheless, there is a high rate of cannibalism of freshly molted isopods (Bedding 1965) thus reducing the chances of survival of the fly larva inside the host and possibly explaining the low prevalence among natural populations.

After the larva has entered the host, it then molts to its 2^nd instar and starts feeding, first on the hemolymph, and then on the organs of the host. The 3^rd instar larva fills most of the body cavity leading to isopod death. Pupation occurs inside the empty exoskeleton of the host (Thompson 1934, Bedding 1965) (Figure 1).

Figure 1. 

Schematic representation of the infection cycle of a Rhinophoridae fly in a woodlouse host. 3 is modified from Thompson (1934).

Almost all records from Rhinophoridae hosts are from the Palearctic region. Outside the Palearctic, there is only mention of Porcellio scaber, Oniscus asellus Linnaeus, 1758 and Porcellionides pruinosus (Brandt, 1833) in the Nearctic (Brues 1903, Jones 1948, Sassaman and Garthwaite 1984, Sassaman and Pratt 1992) and Armadillidium sp. (probably Armadillidium vulgare (Latreille, 1804)) in the Neotropic (Parker 1953). All of these woodlice species were parasitized by Melanophora roralis (Linnaeus, 1758). Nonetheless, all the aforementioned oniscidean and rhinophorid species are introduced from the Palearctic on these locations. Some authors hypothesize that transportation of infected woodlice can explain the occurrence of Palearctic Rhinophoridae in the Nearctic and Neotropic (Mulieri et al. 2010, O’Hara et al. 2015) provided that introduced woodlice are common in these regions (Jass and Klausmeier 2000). The lack of native woodlouse hosts in the Nearctic region is thought to be associated with the low diversity of native woodlice species there (c.f.Schmalfuss 2003), but the same is not true for the Neotropic. In fact, in Brazil alone there is circa 200 described species, most of them native (Cardoso et al. 2018).

In the Neotropic, 19 native species of Rhinophoridae have been described (Cerretti et al. 2014, Nihei et al. 2016), but there is no information regarding parasitoid-host interaction so far. Of these, only the 1^st instar larva of Bezzimyia yepesi Pape & Arnaud, 2001 (Venezuela) is known (Pape and Arnaud 2001) and no host record has been made before, even for the two introduced species, Melanophora roralis (L.) and Stevenia deceptoria (Loew, 1847) (Mulieri et al. 2010) (Figure 2).

Figure 2. 

Distribution map of native and introduced Rhinophoridae species in the Neotropical region (area in gray) including the new larvae record. Apomorphyto inbio records from Pape (2010) and Cerretti et al. (2014); Bezzimyia spp. records from Pape and Arnaud (2001); Shannoniella spp. records from Nihei et al. (2016); Trypetidomima spp. records from Nihei and Andrade (2014); Melanophora roralis records from Parker (1953), Guimarães (1977), González (1998), Cerretti and Pape (2009) and Mulieri et al. (2010); Stevenia deceptoria records from Mulieri et al. (2010). Base map modified from: commons.wikimedia.org/

Here we observed that the Neotropical isopod Balloniscus glaber is a host for the dipterous larvae in southern Brazil (Figure 2), and the two observed 3^rd instar larvae morphotypes are different from the nine Palearctic species with previously described 3^rd instar larval forms (Thompson 1934, Bedding 1965), including the introduced Melanophora roralis.

Balloniscus glaber shares many characteristics with clingers (Wood et al. 2017) although it does not present a typical clinger eco-morphological body type like Porcellio scaber (sensuSchmalfuss 1984). However, it presents clinging behavior (Figure 3A) for predator avoidance (Quadros et al. 2012, Wood et al. 2017) and its legs are shorter than in runner type animals of similar size. These morphological and behavioral characteristics might facilitate larva infection due to reduced distance of sternites to the substrate. Furthermore, like Porcellio scaber, this species also frequently discharges a sticky secretion from their uropod glands upon stimulation (Figure 3B), secretion that is recognized by adult fly females and might stimulate oviposition (Bedding 1965). Five infected individuals have been recorded in the same location (Figure 3C–F). The larvae (one per host) occupied the full body cavity, reaching up to 7 mm in length and resulted in death of all woodlice hosts (Suppl. material 2). Hosts lacked discernible internal reproductive system and the empty gut was the only remaining organ (Figure 3E). No host presented any signs of alteration in overall appearance. The parasitoids could only be identified at the family level due to the lack of larval descriptions for the native species and lack of adults to get a more precise identification. The larvae were identified as Rhinophoridae based on comparative examination of descriptions and illustrations available on the literature; both collected morphotypes presented elongate body shape, anterior and posterior spiracles, and cephaloskeleton as characterized by rhinophorid species. The two 3^rd instar larvae morphotypes are conspicuously different on body shapes, posterior ends, cephaloskeleton, and anterior and posterior spiracles (Figs 4, 5). These forms differ from the known larval stages described by Thompson (1934) and Bedding (1965, 1973). Given the apparent specificity of host records (see next topic) we believe they are Neotropical species (and none of the introduced species). They may be larvae of the described Neotropical species of Shannoniella Townsend, 1939 or Trypetidomima Townsend, 1935, or they may even belong to undescribed species, since the distribution of Balloniscus glaber (Lopes et al. 2005) does not extend to the locations where these native Rhinophoridae have been found, namely, the southeastern portion of Brazilian Atlantic Forest (Nihei and Andrade 2014, Nihei et al. 2016). Furthermore, the location of the new Rhinophoridae record is at a low altitude and Neotropical woodlouse flies seem to be rare in the lowlands, being usually found at elevations of 600–1200 meters in Brazil (Nihei and Andrade 2014, Nihei et al. 2016). Nonetheless, Balloniscus glaber can be found in altitudes up to 1000 meters in southern latitudes (Lopes et al. 2005) while another species from the genus, Balloniscus sellowii (Brandt, 1833), presents a broader latitudinal distribution (Schmalfuss 2003).

Figure 3. 

Balloniscus glaber infected with 3^rd instar Rhinophoridae larva A alive B. glaber clinging to the substrate B secretion discharged from uropod glands C–D dorsal (C) and ventral (D) views of Balloniscus glaber with a third instar Rhinophoridae larva inside E–F partially dissected Balloniscus glaber infected with Rhinophoridae larva showing the empty gut on dorsal view (E) and the calcium plates on ventral view (F).

Figure 4. 

Rhinophoridae larva obtained from the Neotropical isopod Balloniscus glaber. Morphotype 1, 3^rd instar A detail view of the cephaloskeleton on dorsal view B detail view of the anterior spiracle C alive larva with transparent integument D fixed larva, dorsal view.

Figure 5. 

Rhinophoridae larva obtained from the Neotropical isopod Balloniscus glaber. Morphotype 2, 3^rd instar A dorsal view B detail from anterior part showing the cephaloskeleton and anterior spiracles C detail of the posterior spiracle.

A further publication will describe in detail the morphology of the two 3^rd instar morphotypes, and DNA sequencing will be performed trying to obtain a more precise identification.

The earliest reference to a Rhinophoridae parasitoid of woodlice appears to be from von Roser (1840 apudThompson 1934) that created some confusion in the literature in later years. In his paper, the dipteran “Tachinia atramentaria” (currently Stevenia atramentaria (Meigen, 1824)) is mentioned as a parasite of a woodlouse, possibly Oniscus asellus. Thompson (1934), Herting (1961), Bedding (1965) and Verves and Khrokalo (2010) mentioned that Oniscus asellus was probably a wrong identification while Cerretti and Pape (2007) mention Oniscus asellus as a possible host for Stevenia atramentaria. The doubtful record was finally resolved in Kugler (1978) where the author states the record was based on a misidentification of Trachelipus rathkii (Brandt, 1833) according to a personal communication from Herting, that apparently had already been corrected in Sutton’s book (1980). Rognes (1986) and Dubiel and Bystrowski (2016) still list Oniscus asellus as a host or possible host of Stevenia atramentaria but referencing articles that mention the species as a possible host, probably following von Roser’s reference from 1840. Therefore, we could not find any reliable record of Oniscus asellus as a host of Stevenia atramentaria. Dubiel and Bystrowski (2016) list Trachelipus rathkii as a host from Stevenia atramentaria for the first time, but it should be the third record of this interaction if the identification correction from von Roser’s article is taken into account as well as the thesis from Bedding (1965).

The following record on the literature is from Brues (1903) indicating Melanophora roralis as a parasitoid of Porcellio sp., probably Porcellio scaber in Massachusetts, USA. Besides Porcellio scaber as a host, this dipteran species was also recorded as a parasitoid of Oniscus asellus (Jones 1948, Sassaman and Garthwaite 1984, Sassaman and Pratt 1992) and Porcellionides pruinosus (Sassaman and Garthwaite 1984) in the United States. In the Palearctic region, besides the aforementioned isopods, Porcellio spinicornis Say, 1818 (Irwin 1983) is also listed as host for Melanophora roralis. This species of fly shows the highest plasticity of hosts as well as largest geographical distribution that is not restricted to the Palearctic region. It is found in the U.S.A. (Brues 1903, Jones 1948, Sassaman and Garthwaite 1984, Cerretti and Pape 2009), Chile (González 1998), Argentina, Brazil and Jamaica (Guimarães 1977, Mulieri et al. 2010).

Records of interactions are available in von Roser (1840 apudThompson 1934), Brues (1903), Thompson (1934), Jones (1948), Parker (1953)Herting (1961)Bedding (1965), Kugler (1978), Irwin (1983), Sassaman and Garthwaite (1984), Nash (1985), Bürgis (1991, 1992), Cordaux et al. (2001), Wijnhoven (2001), Cerretti et al. (2014) and Dubiel and Bystrowski (2016) although most of this literature is not taxonomically updated. Other works like Arnaud (1978), Draber-Monko (1981), Rognes (1986), Wijnhoven and Zeegers (1999), Cerretti and Pape (2007), and Ziegler (2008) report known hosts from the literature (some of them with current species’ names), but don’t present new records. Therefore, the number of interactions is certainly higher than it has been recorded so far. Currently, there are 18 Isopoda species known to be parasitized (one with an undetermined Rhinophoridae species), and 13 Rhinophoridae species with known hosts, resulting in 35 known interactions (and two others lacking host species identification) and a total of 53 records from 12 countries. Out of the 18 known isopod hosts, only five species have more than one parasitoid: Porcellio scaber (seven or eight rhinophorid species), Oniscus asellus (four spp.), Trachelipus rathkii (three spp.), Armadillidium vulgare (two or three spp.), Porcellio spinicornis and Porcellionides pruinosus (two spp.), and Balloniscus glaber (with two undetermined morphotypes recorded here) (Table 1).