It is well known that some species of Microdontinae (Diptera, Syrphidae) spend their larval and pupal periods in ant nests. Several papers reported that larvae of some microdontine species prey on ant eggs, larvae, and pupae (Van Pelt and Van Pelt 1972, Duffield 1981, Garnett et al. 1985, Reemer 2013, Pérez-Lachaud et al. 2014). At present, 454 microdontine species have been recorded (Reemer and Stahls 2013), and host species for 49 of them have been confirmed as myrmecophilous (Maruyama et al. 2013, Reemer 2013). However, there are few studies on the biology of myrmecophilous microdontine species in Asia (Maruyama and Hironaga 2004).

The microdontine species Microdon katsurai was originally described from Japan (Maruyama and Hironaga 2004). Adult M. katsurai have a remarkable greenish yellow luster. Adult stage M. katsurai are found near nests of the ant Polyrhachis lamellidens Smith 1874 (Hymenoptera, Formicidae) from the end of May to the beginning of July (Hironaga et al. 1998, Katsura 1998, Maruyama and Hironaga 2004). Furthermore, adult females of M. katsurai show ovipositional behavior at the entrance of P. lamellidens nests (Hironaga et al. 1998, Maruyama and Hironaga 2004). These studies imply that P. lamellidens is a host of M. katsurai. However, M. katsurai has never been found inside a P. lamellidens nest, and no interactions between these two species have been observed.

In the present study, to elucidate whether M. katsurai utilizes P. lamellidens, we tried to collect larvae of M. katsurai from a P. lamellidens nest and rear them until they developed into adults.

Collecting samples

We conducted field research on 5 March, 6 March, 15 March, and 16 March 2015 in Hosaka Natural Park (N 35°43'55", E 138°29'18", approximately 600 m in altitude), in the city of Nirasaki, Yamanashi Prefecture, Japan. We found a P. lamellidens nest in several rotten Pinus densiflora Siebold et Zuccarini 1842 trees that had fallen and were piled up (Fig. 1). Each tree was approximately 100 cm in length and 20 to 50 cm in diameter.

Figure 1.  

The nest of Polyrhachis lamellidens in which microdontine larvae and puparia were found.

Three larvae and four puparia of a microdontine were found in the P. lamellidens nest (Figs 2, 3, 4, 5). These larvae were found on a piece of bark in the underground nest of P. lamellidens. Two of the puparia were attached onto a piece of the bark as well, and the other two were found in wood flakes in the P. lamellidens nest. The larva with the pieces of bark were taken and transferred into a transparent plastic container in which wood flakes derived from the P. lamellidens nest were placed on the bottom. All workers and queens of P. lamellidens found in the nest were collected and transferred into a plastic container. All the collected samples were brought to the laboratory.

Figure 2.  

The microdontine larvae and puparia discovered in the P. lamellidens nest. Three larvae and one puparium were attached to pieces of bark.

Figure 3.  

A posterior respiratory process (prp) was observed on a Microdon katsurai larva.

Figure 4.  

A P. lamellidens worker passing by a M. katsurai larva; pl refers to P. lamellidens worker.

Figure 5.  

Puparium of M. katsurai with a P. lamellidens worker; pl refers to P. lamellidens worker.

Figure 6.  

Ventral view of the M. katsurai larva (individual No. 2).

Rearing animals

Morphological studies

Development of a microdontine species under rearing conditions

The microdontine larvae did not show any predatory behavior against P. lamellidens larvae throughout the rearing experiments. Neither was any attack behavior of P. lamellidens workers against the microdontine larvae and pupae observed. One of the larvae (No. 2) started pupation 24 d after collection in the field, and the other two (No. 1 and 3) began pupation 16 d after that (Table 1). Since no exuviae were found in the container prior to pupation of the larvae, the larvae were at the final instar stage at the time they were collected in the field. A pair of discs was observed on the dorsal front part of the individuals (Fig. 7). The appearance of a pair of anterior spiracles from the discs, which is a signature of development from prepupa to pupa, was confirmed 3 d after the beginning of pupation in individual No. 2 and 8 d after the beginning of pupation in No. 1 and 3.

Table 1.

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Days observed for each larva to reach pupal and adult stages after collection of the specimens from their habitat.

Individuals	Pupation	Appearance of anterior spiracle	Emergence of adult
No. 1	16	24	41
No. 2	24	27	48
No. 3	16	24	41

Figure 7.  

A pair of discs on the dorsal front part of the prepupa.

Figure 8.  

A pair of anterior spiracles (as) protruded from the discs.

Individuals No. 1 and 3 emerged as adults 25 d after the beginning of pupation (Table 1, Fig. 9). Both individuals failed to expand their wings, presumably because of a lack of an appropriate substrate for the individuals to mount on and expand their wings appropriately. These two died 2 d after their emergence.

Figure 9.  

A microdontine adult (individual No. 3) with eclosion insufficiency. It has unexpanded wings.

We set a ladder-like scaffold made of a egg paper tray inside a corner of the container for individual No. 2 to mount on 17 d after pupation. In addition, the lid was removed from the container since we noticed that there was condensed water due to the high relative humidity on the inner surface of the container. Then, individual No. 2 emerged with fully expanded wings. The adult of individual No. 2 was transferred into a small glass vial and killed by subjecting it to a temperature of -20ºC in a freezer 6 d after emergence.

Morphology of larval, prepupal, pupal, puparial, and adult Microdontinae

Morphological observations of the microdontine species were conducted at different developmental stages. All the microdontine larvae had a dark brown reticulated structure with a pale green color on the overall dorsal surface (Figs 3, 4). The ventral surface of the larva had an emerald green color (Fig. 6). The whole-body color of the larva changed to light brown when it entered the prepupa stage (Fig. 7). A uniform reticular structure was observed on the dorsal surface of the prepupae and pupae as well as the larvae (Figs 7, 8). The body color of the pupa became dark brown 3 to 8 d after the beginning of pupation (Fig. 8).

Three pieces were made from the puparium shown on the left side in Fig. 10 when the adult emerged. These puparia were 12.8-13.1 mm in length (N = 3), 8.6-8.8 mm in width (N = 3) and 6.0 mm in height (N = 1: the heights of puparia No. 1 and No. 3 were not measured because the dorsal surfaces of these puparia were damaged). The thick marginal band consisted of both dorsal fringe and ventral fringe (Fig. 11). The anterior spiracle on the right side was 398-450 µm in height and 464-488 μm in width (N = 2: the right anterior spiracle of No. 1 was not measured because it was lost at the time emergence) and that on the left side was 410-465 μm in height and 484-497 μm in width (N = 3). In addition, protruding pores were observed on the surface of the anterior spiracles in each individual (Fig. 12). The spiracular opening at the posterior respiratory process was separated into two areas by a flat median carina (Figs 13, 14).

Figure 10.  

A microdontine puparium, dorsal view (individual No. 2). Pieces of the puparium (left) formed after eclosion.

Figure 11.  

Lateral view of the puparium edge (individual No. 2). A dorsal fringe and ventral fringe were observed.

Figure 12.  

Anterior spiracle, lateral view (individual No. 2). ppo refers to protruded pores.

Figure 13.  

Posterior respiratory process, dorsal view (individual No. 2); mcr refers to median carina.

Figure 14.  

Posterior respiratory process, caudal view (individual No. 2).

Concerning the adult, observation was conducted on the specimen casted by individual No. 2 only, which successfully emerged (Fig. 15). The adult of individual No. 2 had a brilliant pale green color with metallic luster except for the caudal part of the abdomen, which was dark purple (Fig. 15). This adult specimen was identified as a female of Microdon katsurai according to Maruyama and Hironaga (2004).

Figure 15.  

Dorsal view of the microdontine adult (individual No. 2).

Based on observations of the appearance and ovipositional behavior of adult M. katsurai near P. lamellidens nests (Hironaga et al. 1998, Katsura 1998, Maruyama and Hironaga 2004), M. katsurai was speculated to spend its immature stages in P. lamellidens nests. In the present study, for the first time, we found M. katsurai larvae from the inside of a P. lamellidens nest and succeeded in rearing them to the adult stage when they were reared with P. lamellidens workers and larvae. This finding provides firm evidence that M. katsurai utilizes P. lamellidens nests. M. katsurai has never been found near nests of other ant species, suggesting that it is a host-specific myrmecophilous species.

P. lamellidens workers were not observed to inspect or attack the M. katsurai larvae in the rearing environment, suggesting that M. katsurai larvae can survive inside a P. lamellidens nest. Howard et al. (1990) demonstrated that the cuticular chemical profiles of the larvae of the myrmecophilous Microdontinae species Microdon albicomatus Novak 1977 is the same as that of its prey, the pupae of the ant Myrmica incompleta Provancher 1881. M. katsurai larvae might also employ such chemical mimicry for avoidance of attacks by P. lamellidens, although no data on cuticular chemical profiles in this species are available. We cannot conclude that the M. katsurai larvae grew by preying on P. lamellidens because they were not observed to prey on or attack P. lamellidens adults or larvae during the rearing experiments. Nevertheless, all the M. katsurai larvae pupated and emerged (Table 1). Thus, it is obvious that these larvae had fed on some food source sufficient for them to grow into adults before we had collected them. It has been reported that larval microdontines prey on ant eggs, larvae and pupae (Van Pelt and Van Pelt 1972, Duffield 1981, Garnett et al. 1985, Reemer 2013, Pérez-Lachaud et al. 2014). Others have suggested that the larvae of certain Microdontinae feed on refuse or pellets of food ejected by ants (e.g. Wheeler 1908), but such feeding behavior has never been confirmed (Reemer 2013). Further studies in both the field and laboratory are required to reveal the types of food resources M. katsurai larvae utilize.

Having been collected in March, the stage of all the larvae was likely the last instar because they did not molt before becoming pupae. Adult M. katsurai appear from the end of May until the beginning of July (Hironaga et al. 1998, Katsura 1998, Maruyama and Hironaga 2004), suggesting that this species overwinters in an ant nest before pupation. The three individuals studied emerged between 25 April and 2 May 2015 under laboratory rearing conditions. The time of emergence in the present study was almost one month earlier than the recorded observations for adults of this species in the field (Maruyama and Hironaga 2004). This difference in appearance time may be due to the rearing temperature (25ºC) in the present study, which is higher than that of the natural habitat of this species.

The color of larvae of microdontine species is white or brown (Wheeler 1908, Wheeler 1924, Greene 1955, Dixon 1960, Rotheray 1991, Maruyama et al. 2013, Schmid et al. 2014). The pale green color shown on the dorsal surface of the M. katsurai larvae in this study is rare in Microdontinae; the larvae of most species in this subfamily show a white or brown color (Wheeler 1908, Wheeler 1924, Greene 1955, Dixon 1960, Rotheray 1991, Maruyama et al. 2013, Schmid et al. 2014). This is the first record in Asia of a microdontine larva with such a remarkable color.

Based on morphological studies of adult specimens, M. katsurai is classified into the subgenus Chymophila (Reemer and Stahls 2013). Our morphological studies on the M. katsurai puparium support this classification. There are several morphological similarities between the M. katsurai puparium and that of Microdon (Chymophila) fulgens, such as dorsal surfaces that have a uniform reticular structure, a thick marginal band consisting of both a dorsal fringe and a ventral fringe, and anterior spiracles with a height that is shorter than its width (Thompson 1981). Moreover, in the puparium of both species, a flat median carina divides the two areas of the opening of the spiracles at the spiracular plate of the posterior respiratory process, which is characteristic in the subgenus Chymophila (Thompson 1981). Considering all this together, the classification by puparium morphology in the present study is concordant with that of the adult morphology.

The two individuals that failed to emerge, No. 1 and No. 3, could not be identified using adults, but they are considered to be M. katsurai based on the morphology of their puparia.

In Japan, thus far, M. katsurai has been recorded in the Nagano, Ibaraki, Tochigi, Mie, Osaka, Hyogo, Yamaguchi, Kagawa and Kagoshima prefectures (Hironaga et al. 1998, Katsura 1998, Kano 1999, Tanaka 2002, Kusama and Tamaki 2004, Genka 2010, Iwai 2010, Sadahiro 2013, Noishiki 2014), and for the first time, it was collected in the Yamanashi prefecture in the present study. Although M. katsurai is often recorded in lowland environments (Maruyama and Hironaga 2004), it has also been found in habitats with relatively high altitudes of up to 600 m (Noishiki 2014). The altitude of the habitat where M. katsurai was found in the present study is approximately 600 m, suggesting that the habitat preference of this species is broader than previously speculated.

M. katsurai is a rare species that is designated as Vulnerable (VU) in Japan (Ministry of the Environment 2015). Furthermore, the habitat of P. lamellidens, which is designated as Vulnerable (VU) as well, is decreasing from the influence of recent residential and industrial development (Maruyama and Hironaga 2004, Maruyama et al. 2013, Sadahiro 2013, Ministry of the Environment 2015). The present study indicated a strong ecological association between M. katsurai and P. lamellidens. It is necessary to protect the P. lamellidens habitats immediately to conserve not only P. lamellidens but also the very rare species M. katsurai.

The authors thank Mr. T. Isoda from Tokyo University of Foreign Studies and Mr. Y. Takeshita from Nihon University for helping to collect samples. We also thank Dr. N. Kono from Keio University for providing useful advice on this study. This research was supported in part by research funds from the Yamagata Prefectural Government and Tsuruoka City, Japan.

The authors declare that there are no conflicts of interest.