Reproductive behavior and early immature morphology of Portschinskia magnifica: implications for evolutionary biology in bot flies (Diptera: Oestridae)

Abstract

Portschinskia Semenov is a rare genus of bot flies whose larvae are obligate parasites of pikas and murine rodents, crucial for understanding the evolutionary biology of Oestridae. However, limited information on their adult biology and early immature stages has hindered the progress. Here, we provided the first documentation of adult oviposition, behavior, and morphology of newly hatched first instars of P. magnifica Pleske. Using confocal laser scanning microscopy and scanning electron microscopy, we characterized the ultrastructure of egg and first-instar larva, identifying key traits facilitating attachment. Eggs were deposited individually or in groups, and glued on non-host surfaces with white adhesive substances, without specialized attachment organ. Newly hatched first instars were circled with an anterior spinose band on each body segment except the anal division, awaited hosts in an upright position, and anchored to the egg shell by their anal division, likely supported by curved spines originating from the peritreme of the posterior spiracles. Evolutionary analyses of reproductive behavior across the 4 bot fly subfamilies reveal at least 3 times independent evolution of oviposition on non-host surfaces in Portschinskia or the Hypodermatinae clade as a sister group to Ochotonia, Gasterophilus pecorum (Fabricius) (Gasterophilinae), and Cuterebrinae. In contrast, species in the Oestrinae are larviparous, depositing first instars directly onto hosts. Our findings shed light on oviposition behavior and early immature morphology of the rare genus Portschinskia, offering insights into reproductive strategies and evolutionary adaptations of bot flies.

Bot flies (Oestridae) have attracted significant attention as their larvae are causatives of myiasis in humans, livestock, and wild mammals, with hosts ranging from elephants to rodents (Zumpt 1965; Colwell et al. 2006). Despite being a relatively small family with ~170 species classified into 4 subfamilies (Cuterebrinae, Gasterophilinae, Hypodermatinae, and Oestrinae) (Pape 2001; Li et al. 2020b), oestrids exhibit remarkable diversification in biology and morphology (Zumpt 1965; Li et al. 2018). They are either oviparous or larviparous, with females depositing variously shaped eggs or first-instar larvae on host animals or on non-host substrates. First-instar larvae of bot flies enter hosts through dermal or mucosal penetration and develop in subcutaneous, nasopharyngeal, or intestinal sites (Catts 1982; Colwell et al. 2006). Adult oestrids are short-lived with vestigial mouthparts, but many are characterized by large body size, dense pilosity, and contrasting warning colouration, resembling aculeate bees in a notable example of mimicry (Sabrosky 1986; Nilssen et al. 2000; Colwell et al. 2006; Li et al. 2020a). Despite extensive studies on species parasitizing domestic animals, there is limited information about the adult biology and early immature stages of other species in Oestridae (Zumpt 1965; Colwell et al. 2006). This has posed an obstacle to furthering our understanding of the life-history strategies and evolution of these parasitic flies.

Reproductive behavior is of great importance for oestrid adaptations, as it directly impacts larval survival and the overall fitness of these parasites (Colwell et al. 2006; Refsnider and Janzen 2010). Oviposition site selection is highly specific among bot flies. For instance, some female oestrids are known for their persistence in ovipositing on hosts (Colwell et al. 2006). A notable case is Gasterophilus intestinalis (De Geer), which has been observed actively pursuing galloping horses and resuming egg laying as soon as the host stops (Cope and Catts 1991). After hatching, the first-instar larvae penetrate the host near the egg site, facilitating quick entry into the host’s body (Zumpt 1965). In contrast, species in Cuterebrinae glue their eggs on grass blades or stones (Catts 1982), with newly hatched larvae adhering to passing hosts that touch them (Catts 1967). It has been hypothesized that oviposition site selection has undergone strong evolutionary pressures to maximize offspring survival (Resetarits 1996; Fenton and Rands 2004; Refsnider and Janzen 2010). However, the evolutionary history of bot fly oviposition habits remains unclear due to the lack of relevant information on Portschinskia Semenov (Pape 2001, 2006).

The genus Portschinskia represents a key group for advancing our knowledge of bot fly evolution. It diverges from the genus Ochotonia and forms a sister–group relationship with the other genera within the Hypodermatinae subfamily (Pape 2001; Pape et al. 2017). With 11 described species, they are very rare in collections (Li et al. 2020a), resulting in a shortage of morphological and biological information. Adult Portschinskia exhibits startling mimicry of bumblebees, while their larvae are subcutaneous parasites, developing within pikas and murine rodents (Grunin 1965; Li et al. 2020a). Eggs dissected from gravid females of Portschinskia loewii Schnabl lack an attachment organ and displayed concave ventral surfaces (Grunin 1965), suggesting they may be glued to either host or non-host surfaces (Pape 2006). To date, no data are available on the female oviposition and the newly hatched larval behavior in any Portschinskia species. Eggs and first instars still need to be morphologically characterized in detail.

This study aims to address this gap through a study of P. magnifica. We provide the first documentation of oviposition on non-host substrates and the “ambushing” host-finding behavior of newly hatched first instars in Portschinskia. Using scanning electron microscopy and confocal laser scanning microscopy, general and ultrastructure of the egg and first-instar larva is characterized, identifying key traits for attachment. We discuss the variation in oviposition behavior of bot flies from an evolutionary perspective, revealing that oviposition on non-host surfaces has arisen at least 3 times in Oestridae. Our findings enrich morphological and biological knowledge of the cryptic genus Portschinskia and provide valuable insights into the adaptive evolution in bot flies.

Materials and Methods

Sample collection and behavioral observations

Adult specimens of P. magnifica (Figure 1A, B) were observed in Songshan National Nature Reserve, Beijing, China (115°43ʹ44″E, 40°29ʹ9″N) during July in each of the years 2016–2020. Specimens were collected using a standard insect sweep net. Sixteen live adults were subsequently brought to the laboratory of Beijing Forestry University (Supplementary Table S1). For behavioral observations (conducted in 2016–2017), a male and female pair, or a single female, were placed in a standard plastic box (20 cm × 20 cm). The box was covered with finely perforated cloth and lined with 2 layers of tissue paper on the bottom. Fresh leaves (not identified) were supplied for humidity and as a natural substrate. Newly deposited eggs were transferred to a climate box and incubated under 50% humidity, 25/14 °C and 12/12 h day/night. A number of emerging first-instar larvae were killed by soaking in near-boiling water (~95 °C) and stored in 70% ethanol. Infections of laboratory mice were done by picking up newly hatched first instars with the tip of an insect pin and gently putting them down on the back of a mouse. Five wild-caught Apodemus peninsulae (Thomas) infected with P. magnifica larvae were also found during the years of field work (Figure 1C, D; Supplementary Table S2). All the experiments strictly followed the Guidelines for Animal Experimentation of Beijing Forestry University. Infected mice were inspected daily by manual examination for the presence and condition of warbles (Figure 1E–G). In addition, both hatched and unhatched eggs were sampled from the insect box for morphological examination.

Figure 1.

Portschinskia magnifica Pleske, mating and host infection. A. Adult male (left) and female (right); B. Mating behavior of a male and a female P. magnifica; C–D. Wild-caught Apodemus peninsulae (Thomas) (Rodentia: Murinae) infected with larvae of P. magnifica on left side; E–G. Laboratory mice infected with larvae of P. magnifica.

Sample preparation and imaging

For scanning electron microscopy, the first instars were dehydrated through an ascending series of ethanol (85%, 90%, 95%, and 100% ethanol; 10–30 min each), critical-point dried in CO₂, mounted on aluminum stubs, coated with platinum, and finally examined using a HITACHI SU8010 (HITACHI, Tokyo, Japan) scanning electron microscope. Eggs were mounted and coated similarly but without going through dehydration. Endochorion was exposed by dissecting the naturally hatching eggs with fine anatomical forceps. For confocal laser scanning microscopy, larvae were cleared with 10% KOH at room temperature (2–6 h), rinsed in dH₂O, and slide-mounted in 50% glycerin solution. Samples were examined using a ZEISS LSM780 (ZEISS, Oberkochen, Germany) confocal laser scanning microscope. Three different lasers were used (wavelengths: 405 nm, 488 nm and 633 nm) and autofluorescence signals were collected in 3 PMT channels: 410–468 nm (blue), 508–534 nm (green), and 644–759 nm (red). Maximum intensity projection (MIP) stackings were produced using Fiji or Zeiss imaging software (ZEN). Morphological terminology follows Hinton (1981) and Grzywacz et al. (2012) for eggs, Szpila et al. (2014, 2017) and Li et al. (2021) for first instars, and Cumming and Wood (2017) for adults. Voucher specimens have been deposited at the Beijing Forestry University.

Ancestral state reconstruction

To trace the evolutionary history of reproductive behavior in bot flies, we used ancestral state reconstruction (ASR) methods implemented in Mesquite 3.51 (Maddison and Maddison 2018) and RASP 4 (Yu et al. 2020). We examined character evolution across bot flies, employing the most recent and well-supported genus-level cladogram of Oestridae from Pape et al. (2017) as the backbone phylogeny. This cladogram was constructed based on 124 morphological characters, providing high resolution and strong Bremer support for the main branches (Pape et al. 2017). We classified 2 dominant types of reproductive behavior: females either depositing eggs or first-instar larvae on food sources or host animals, or on non-host substrates. All characters were scored and compiled from the literature and our filed observations, with missing data indicated by question marks. The species used for character coding are listed in Supplementary Table S3. For ASR, we utilized maximum parsimony (MP) and maximum likelihood (ML) methods in Mesquite 3.51. The MP approach minimizes the number of evolutionary steps required to explain the dataset (Herbst and Fischer 2017). The ML analysis utilized the Markov k-state 1 parameter model (Mk1), which assumes equal probabilities for character state changes (Lewis 2001). In addition, we conducted the Bayesian Binary method (BBM) analysis (Ronquist and Huelsenbeck 2003; Ali et al. 2012) in RASP 4 (Yu et al. 2020), using default parameters for ASR.

Results

Biological behavior of adults and newly hatched larvae of P. magnifica

Our study showed that females of P. magnifica oviposit on non-host substrates (Figures 1 and 2). The eggs were deposited on tissue paper and fresh leaves, either in groups or individually on the upper surface or margin, and they were glued with a white adhesive substance at their posterior end and oriented perpendicular to the surface (Figure 2A–C). The first-instar larvae spontaneously hatched after about a week, emerging from the eggshell through a slit in the anterior end but with the anal division remaining inside. Initially, the larva stayed motionless with its body almost in straight continuation of the longitudinal axis of the egg. Vibrations or gentle blowing would cause the larva to initiate energetic swinging movements, gripping onto any nearby object, including human fingers. When presented to a mouse, first instars readily moved from the tip of an insect pin onto the mouse, eventually disappearing from the observer’s sight. After 13–15 days, subcutaneous warbles containing a single larva were easily detectable, often located on the posterior part of the host’s abdomen, with the breathing hole facing towards the base of the host’s tail (Figure 1F, G). Occasionally, warbles developed on the head (Figure 1E). High larval numbers were observed in natural hosts, as noted by Grunin (1965), who reported a single Apodemus sp. host infected with 15 larvae in various development stages.

Figure 2.

Portschinskia magnifica Pleske, egg and first-instar larva. A–C. Eggs deposited on leaves; C–E. Eggs with first-instar larvae anchored to the egg shell by their anal division. [All images from observations in the laboratory.]

Morphological characterization of egg and first-instar larva of P. magnifica

Egg (Figures 2 and 3): Length 0.76–0.79 mm, width at middle 0.17–0.19 mm (n = 10). Yellow in color. Elongated ovoid with rounded poles, the anterior pole more blunted; ventral surface flattened or slightly concave. Chorionic sculpturing predominantly featured with reticular patterns of irregular pentagons and hexagons. Hatching pleat developed as an apical suture, continuing laterally along the anterior 1/7–1/6 of the egg. Micropyle recessed into the anterior pole, and irregular-sized aeropyles distributed along the dorsal rim of the hatching pleat (Figure 3D). Endochorion developed as a fine meshwork of struts and tubercles (Figure 3E, F).

Figure 3.

Portschinskia magnifica Pleske, egg. A. Habitus, ventral view; B. Chorion, sculpturing of ventral surface; C. Habitus, lateral view; D. Egg, anterior part after hatching of larva; E, F. Endochorion meshwork near hatching suture. Scale bars: A = 250 μm; B = 25 μm; C = 200 μm; D = 50 μm; E, F = 5μm.

First-instar larva (Figures 4–7): Body fusiform (Figures 4 and 5A), composed of a small bilobed pseudocephalon (pc), 3 thoracic segments tI–tIII, 8 abdominal segments aI–aVIII, and an anal division (ad) bearing the posterior spiracles. Each segment was encircled by an anterior spinose band, consisting of up to 4 irregular rows of spines, except for the anal division, where spines were found only around the spiracular plates.

Figure 4.

Portschinskia magnifica Pleske, first-instar larva, general morphology. A. Habitus, dorsal view. B. Habitus, lateral view. C. Habitus, ventral view. D. Pseudocephalon and thoracic segments, ventral view. D. Pseudocephalon and thoracic segments, lateral view. F. Anal division, dorsal view, showing the peritreme of the posterior spiracles. Abbreviations: aI–aVII = abdominal segments I–VIII, ad = anal division, pc = pseudocephalon, tI–tIII = thoracic segments I–III. Scale bars: A–C = 0.1 mm; D–E = 0.05 mm; F = 20 μm.

Figure 5.

Portschinskia magnifica Pleske, first instar larva, with maximum intensity projection stackings. A. Habitus, dorsal view. B. Pseudocephalon and tI–III, dorsal view. C. Cephaloskeleton, dorsal view. D. Anal division and aVIII, dorsal view. E. Anal division, with depth coding. Abbreviations: aI–aVII = abdominal segments I–VIII, ad = anal division, lb = labrum, mh = mouthhook, pc = pseudocephalon, tI–tIII = thoracic segments I–III. Scale bars: A = 100 μm; B = 50 μm; C = 20 μm; D = 40 μm.

Figure 6.

Portschinskia magnifica Pleske, first-instar larva. A. Pseudocephalon and thoracic segment I, anterior view; B. Pseudocephalon and thoracic segments I–II, dorsal view; C. Pseudocephalon and thoracic segment I, ventral view; D. Antennomaxillary sensory complex, anterior view; E. Maxillary palp; F. Oral area, anterior view; G. Left mouthhook; H. Pseudocephalon and thoracic segment I, dorsal view; I. Rounded and tapered dorsal spines and a sensillum coeloconica on tI; J. Plaque-like sensillum coeloconica on tI. Abbreviations: and = antennal dome, ap = additional pit, as = additional sensillum, mh = mouthhook, mp = maxillary palp, sb = sensilla basiconica, scI = sensilla coeloconica I. Scale bars: A = 15 μm; B = 50 μm; C = 20 μm; D = 2.5 μm; E = 1 μm; F, G = 5μm (inset = 2 μm); H = 10 μm; I = 2 μm; J = 1.5 μm.

Figure 7.

Portschinskia magnifica Pleske, first-instar larva. A. The first thoracic segment, ventrolateral view, showing body spines; B. The first thoracic segment, ventral view, body spines and sensilla (inset: coeloconic sensillum); C–E. Posterior spiracular plate (inset: coeloconic sensillum); F–G. Coeloconic sensilla of posterior spiracular plate; H. Spines and coeloconic sensillum around posterior spiracular plate; I. Coeloconic sensillum of posterior spiracular plate. Abbreviations: scI–V = sensilla coeloconica I–V. Scale bars: A, C = 15 μm (inset = 3μm); B = 2.5 μm (inset = 2 μm); C = 15 μm; D, H = 5 μm; E = 10 μm; F = 1.5 μm; G = 2 μm; I = 1 μm.

Pseudocephalon (Figures 4A–E, 5A–C, and 6) with paired antennomaxillary sensory complex and each composed of an antennal dome (and) and a maxillary palpus (mp). Antennal dome (Figure 5D) circular, distinctly elevated from the surrounding cuticle, with a mesh of micropores on the dorsal surface. Maxillary palpus (Figure 5D, E) with 2 equal-sized sensilla basiconica (sb), each composed of a large peg with a flat surface, and 3 sensilla coeloconica (as) sunken into deep pits.

Cephaloskeleton (Figures 4D–E, 5C, and 6F, G) with mouthhooks conspicuously protruding from the oral aperture, well developed, strongly sclerotized, subrectangular apart from the curved, pointed tip, and with a longitudinal concavity or groove laterally on the distal half. Labrum is strongly sclerotized, small and triangular (Figure 5C). Parastomal bars longer than broad; intermediate sclerite with dorsal cornu longer than ventral cornu.

Thorax (Figures 4A–C, 5A–C, and 6A–C, H) with first segment (tI) composed of a collar of the densely set, prominent spinose band (Figures 4A, B and 5A–C); The second segment (tII) with irregular rows of triangular spines ventrally (Figures 6C and 7A) and a sclerotized, dome-like plate dorsally (Figures 5A, B and 6B). The third segment (tIII) with 3 to 4 irregular rows of spines both dorsally and ventrally. Posterior to the spinose band, located pairs of sensilla basiconica, one with a large peg with a flat surface and one with a slender, protruding peg (Figure 6H–J). Keilin’s organ not developed.

Abdomen (Figure 5A) with each of aI–aVII distinctly more than twice as broad as long; aI–aVI encircled by 3 or 4 irregular rows of broad-based spines with pointed tip; aVII with few spines medially; aVIII with no spines medially. Each of aI–aVII dorsally with several large pegs with a flat surface similar to those on the thoracic segments (Figure 6H–J).

Anal division (Figures 4F, 5A, D, E, and 7C–I) with spinose band confined to the cuticle around the spiracular plates. Coeloconic sensilla around spiracular field (scI–VI) with raised collar and rim bulged in one or more short projections (Figure 7F–I). Peritreme of each spiracle produced into 2 strong spines medially: a long, tapering, posteriorly directed spine ventro-medially, and a slightly shorter, recurved spine dorso-medially (Figures 5A, D, E and 7C–E). One or 2 small additional tubercular processes are also present on the peritreme.

Remarks.

Grunin (1965) mentioned the presence in the second instar of a cap-like structure (“die Haube”) dorsally on what he considered the first thoracic segment. This is probably the same structure as the sclerotized, dome-like plate taking up most of what is here considered the dorsal surface of tII (Figure 5A, B). Grunin (1965, fig. 106) illustrated the peritremes of the first-instar posterior spiracles of P. magnifica with their adornment of spines, but he did not comment on their possible function.

Ancestral state reconstruction of reproductive behavior in bot flies

Our ancestral state reconstruction of reproductive behavior using BBM, MP, and ML analyses revealed similar evolutionary patterns across bot flies (Figure 8; Supplementary Figure S1). The results suggest that the ancestral condition for bot flies was likely to deposit offspring directly onto the host. Transitions between deposition on hosts and non-host substrates occurred throughout the Oestridae family. Specifically, our analysis revealed that oviposition on non-host surfaces has independently evolved at least 3 times across the 4 oestrid subfamilies: in the Cuterebrinae, in Gasterophilus pecorum (Fabricius) (Gasterophilinae), and in the Hypodermatinae clade as a sister-group to Ochotonia, as shown by BBM analysis (Figure 8). Similarly, MP and ML analyses support the evolution of this behavior in the Cuterebrinae, in G. pecorum and in Portschinskia within Hypodermatinae (Supplementary Figure S1). In addition, the ML analysis was limited in confirming ancestral states for Gasterophilinae and Hypodermatinae due to insufficient data from Ruttenia, Neocuterebra, and Ochotonia.

Figure 8.

Ancestral state reconstruction of offspring deposition site in Oestridae using Bayesian binary MCMC analysis (BBM) in RASP 4. The most likely ancestral state is noted by a letter in the center of each pie diagram, with alternative states coloured according to their likelihood proportions. Cladogram used for reconstruction follows the morphology-based phylogeny in Pape et al. (2017).

Discussion

Variation in oviposition site selection is important for understanding life history evolution (Resetarits 1996), as it has been subject to strong selection pressures to maximize offspring survival (Refsnider and Janzen 2010). Evaluation of the ancestral oviposition habit of the bot flies has long been limited due to insufficient information on Portschinskia. Under laboratory conditions, we observed the female of P. magnifica depositing eggs on non-host substrates, providing the first evidence of ovipositional behavior in this genus. Although caged insects may display unnatural behavior, we consider that our observations closely reflect the behavior under natural conditions. For this discussion, we assume that this behavior is representative of the entire genus. Optimizing oviposition site selection based on the most recent phylogeny of Oestridae (Pape et al. 2017) suggests that the ancestor of bot flies deposited offspring directly on the host. The behavior of oviposition on non-host substrates has independently evolved at least 3 times, in Portschinskia or the Hypodermatinae clade as a sister group to Ochotonia, G. pecorum (Fabricius), and Cuterebrinae (Figure 8). However, the ancestral state of oviposition biology for Hypodermatinae and Gasterophilinae remains unresolved due to the lack of information from the basal genera Ochotonia Grunin (monotypic, known only from a single third instar larva) (Grunin and Vazhev 1968; Pape 2001; Pape et al. 2017) and Neocuterebra Grünberg and Ruttenia Rodhain [subcutaneous parasites of the African forest elephant, Loxodonta cyclotis (Matschie)] (Zumpt 1965). Further studies on the adult biology of these rare and cryptic bot fly genera are in demand to better understand the evolution of oviposition behavior in Oestridae.

Bot flies exhibit various modes of reproduction (Zumpt 1965; Colwell et al. 2006). Apart from the larviparous females in Oestrinae, bot flies from the other 3 subfamilies are oviparous, with females depositing eggs on host animals or non-host substrates (Anderson 2006). Species in Cuterebrinae typically glue eggs on grass blades or stones (Catts 1982), whereas the females in Hypodermatinae and Gasterophilinae predominantly attach their eggs firmly to host hairs (Zumpt 1965; Anderson 2006). Our observations represent the second documented case of oviposition on non-host substrate for a non-cuterebrine bot fly. The only other known case is the gasterophiline species G. pecorum, which oviposits on grass blades and other low vegetation nearby equid hosts (Grunin 1969; Huang et al. 2021). In addition, a noteworthy difference between P. magnifica and both G. pecorum and the Cuterebrinae is the absence of a specific attachment organ on the egg (Grunin 1965; Pape 2006; present study). Previous studies demonstrated that bot fly eggs are attached to host or non-host substrates through a unique structure located either on the ventral terminus or the ventral surface of the eggs (Cogley 1991; Colwell et al. 1999; Li et al. 2019). In contrast, our evidence indicates that P. magnifica eggs are attached solely by a sticky substance at the posterior pole of the egg, representing another approach to egg attachment in Oestridae.

First-instar larvae of P. magnifica exhibit a passive “ambushing” (sit-and-wait) strategy for infection, which appears to be a result of convergent evolution. Newly hatched larvae of P. magnifica stand erect, anchored in the eggshell, and swing their anterior end to make contact with a potential host when stimulated. This strategy of waiting for a passing host and the agitated behavior of the first instar when sensing a potential host (e.g., through vibrations, air movements, or a rise in temperature or CO₂ concentration) is similar to behaviors observed in species of Cuterebra Clark (Catts 1982), Rhinophorinae (Bedding 1973; Pape and Arnaud 2001) and some species in Calliphoridae [e.g., Cordylobia anthropophaga (Blanchard and Bérenger-Feraud)] (Zumpt 1965). Furthermore, the paired and pointed cuticular structures on the peritreme of the posterior spiracles in first-instar P. magnifica are well-suited for gripping the endochorionic meshwork, facilitating the anchored position of the larva. A similar adaptation has been documented in the only other species of Portschinskia, for which the first instar is known [possibly P. loewii (Schnabl), see Grunin 1965: figs 85, 86], but not in any other bot fly species, suggesting this may be a unique ground-plan feature of Portschinskia. In addition, modifications on the first-instar anal division—such as an adhesive sac in species of Cuterebra (Baird and Graham 1973) and ventral cuticular ridges or adhesive vesicles in the Rhinophorinae (Bedding 1973; Cerretti et al. 2020)—serve a similar purpose of anchoring the first instar in the eggshell or to the substrate while maintaining an “upright” position. However, these structures are not derived from the peritreme and therefore not considered homologous with the condition in Portschinskia. This may support that the “ambush” strategy of infecting suitable hosts has evolved convergently in Portschinskia and Cuterebra.

Migration behavior of the first-instar larva varies among bot flies, with some species undergoing extensive migrations within their hosts, while others develop at the initial site of penetration (Grunin 1965; Zumpt 1965; Colwell et al. 2006). Our observation does not provide conclusive evidence on whether the first instar of P. magnifica reaches the site where the warble develops through subdermal migration. In Hypodermatinae, extensive migration within host tissues has been reported in some species, particularly those parasitizing ungulates. Grunin (1965) documented the position of warbles produced by rodent-parasitizing species of the genera Oestroderma Portschinsky and Oestromyia Brauer, noting a close match to the position of eggs glued to host hairs and implying non-migration behavior. However, it has been documented that the first instar of Oestromyia leporina (Pallas) migrates subcutaneously for 3–4 days in the common vole Microtus arvalis (Pallas) (Rietschel 1975). Larvae of Portschinskia appear to produce warbles in laboratory mice in a position similar to those found in hosts of Oestroderma and Oestromyia. A similar migration seems likely for P. magnifica, considering that first instars were applied to the back of the hosts, and warbles were discovered about 2 weeks later during daily examinations. Alternatively, the concentration of P. magnifica larval warbles on the posterior part of the host abdomen might result from the ability of the first instar to actively crawl from the dorsum down to the abdominal area, searching for a favorable position to penetrate the skin and form the warble. The rare occurrence of warbles on the head could be caused by a shift to the nose of another host through grooming or other contact behaviors.

Overall, our findings provided the first documentation of reproductive behavior and early immatures of P. magnifica and suggested that a passive “ambushing” host behavior of newly hatched first instars of P. magnifica appears to be a result of convergent evolution with a similar behavior in the genus Cuterebra (Catts 1982). We characterize the ultrastructure of egg and first-instar larvae and revealed key characters facilitating egg attachment and newly hatched larvae anchored in the eggshell. In addition, the evolutionary history of oviposition behavior was reconstructed across all 4 oestrid subfamilies, with evidence supporting the independent evolution of oviposition on non-host surfaces in Portschinskia, as well as in G. pecorum and Cuterebrinae. Collectively, our results enriched morphological and biological knowledge of the cryptic oestrid genus Portschinskia, which will facilitate future studies on the phylogenetics and evolutionary diversifications of bot flies.

Supplementary Material

Supplementary material can be found at https://academic.oup.com/cz.

Authors’ Contributions

The study was conceived by X.Y.L., D.Z., and T.P. Data collection was led by X.Y.L., with contributions from W.T.X., D.Z., Y.Q.F., and T.P. X.Y.L., W.T.X., and T.P. performed the analyses, with contributions from D.Z. The original draft of this manuscript was written by X.Y.L., W.T.X., and T.P. and revised by all authors.

Funding

This study was supported by the National Natural Science Foundation of China (grant numbers 32170450 and 31872964) and Science & Technology Fundamental Resources Investigation Program (grant number 2022FY202100).

Conflict of Interest

The authors declare no competing interests.