Ephemeroptera.

Two adult mayflies, Ephemera sp. (Ephemeridae) and Neocloeon triangulifer (Baetidae), both females, were found to possess the inflated alimentary canal, a feature first noted for mayflies in general by Swammerdam [28] in the earliest microscopic dissections of insects. Both adults possess a large, elongate inflated space, beginning near (Ephemera) or inside (Neocloeon) the head capsule, proceeding through the thorax and extending through the 7^th abdominal segment (Fig 1A and 1B). The inflated space is constricted slightly in the thorax, likely where wing and leg muscles are positioned. An additional constriction is also present in Ephemera in the 4^th and 5^th segments before expansion into the final two segments of the air space; this constriction is not present in Neocloeon.

A subimago female Neocloeon triangulifer (Baetidae) was found to possess a partially inflated alimentary canal (Fig 3), extending in three parts from the posterior of the head capsule into the 3^rd abdominal segment.

Download:

• PNG
  larger image
• TIFF
  original image

Fig 3. Partially filled gut in Neocloeon triangulifer (Ephemeroptera: Baetidae).

Note dimpled appearance in posterior air space, possibly caused by presence of eggs. Scale bar 1 mm.

https://doi.org/10.1371/journal.pone.0351543.g003

Plecoptera.

An adult female Isoperla (Perlodidae) was found to have a short but pronounced inflated gut space, beginning in the mid-thorax and extending through the anterior part of the abdomen (Fig 2A). This specimen was also found to have a substantial number of eggs in various stages of development (Fig 4). We hypothesize that the inflated alimentary space in this specimen is shortened to accommodate the growth and development of the eggs.

Download:

• PNG
  larger image
• TIFF
  original image

Fig 4. Eggs in abdomen of Isoperla sp. (Plecoptera: Perlodidae).

Medial longitudinal X-ray section showing position of eggs in abdomen of Isoperla, just posterior from air space. (See inset for position of slice.).

https://doi.org/10.1371/journal.pone.0351543.g004

Embioptera.

The adult male Oligotoma nigra webspinner (Oligotomidae) possesses a very long inflated cavity, beginning approximately halfway into the head capsule, just behind the eyes, and extending nearly the length of the body (Fig 2B). Several small constrictions were observed in the abdominal section of the inflated space.

Megaloptera.

An adult Corydalus cornutus (Corydalidae) female features a very large, inflated space, notably filling much of the head capsule (Fig 2C). The size of the head air space is such that it is possible the pharynx fuses with other head air sacs during development. The air space extends posteriorly through the thorax and into the abdomen, where it extends laterally almost to the inner body wall. Constrictions in the thorax are likely the result of large, paired flight muscles positioned dorsoventrally in the thorax.

Strepsiptera.

The adult Xenos peckii (Xenidae) male possesses an inflated space beginning in the second thoracic segment, extending posteriorly to the anterior portion of the abdomen (Fig 1C). Slight constrictions in the thoracic portion of the air space are likely due to the presence of flight muscles.

Lepidoptera.

The adult female Bombyx mori (Bombycidae) silkworm moth (domestic) shows a pronounced abdominal air space (which may be the crop), extending from the posterior portion of the thorax into the anterior of the 4^th abdominal segment (Fig 2D). The silkworm moth was the largest specimen found to have an inflated alimentary space, and while not as large relative to the rest of the body as the other insects, the known lack of adult feeding suggests possible co-opting of the digestive tract.

Diptera.

The male parasitoid spider fly Ogcodes sp. (Acroceridae) features an extreme example of inflated alimentary space, filling nearly the entire abdomen (Fig 1E). Indeed, the air volume comprises approximately 50% of the entire volume of the insect. The air space is bilobed (Fig 5), indicating that the crop, occasionally filled with air in Diptera during digestion [29], has been completely inflated in addition to the remainder of the alimentary canal.

Download:

• PNG
  larger image
• TIFF
  original image

Fig 5. Inflated gut of spider fly Ogcodes sp. (Diptera: Acroceridae).

Dorsal view showing bilobed nature of inflated alimentary space (including crop), filling nearly the entire abdomen. Scale bar 1 mm.

https://doi.org/10.1371/journal.pone.0351543.g005

Ephemeroptera.

Mayflies feed on a variety of food sources during the aquatic immature stage; while most species feed on algae and other organic matter in the water, others are carnivorous and eat other invertebrates [1]. Rather than being absent or reduced in size, adult mayflies typically possess the full complement of mouthparts, however they are weakly sclerotized and clearly non-functional for feeding [30].

Plecoptera.

Stonefly immatures have a varied diet; some are detritivores, feeding on decaying plant, fungal, and algal matter [31], while others are omnivorous or carnivorous. Ontogenetic changes in feeding strategy between earlier and later instars can occur in many species [32,33]. The adult female Isoperla specimen included here (Fig 7A) retains partially reduced mouthparts, a characteristic of the infraorder Systellognatha [33,34], which indicates that it likely does not feed on solid material.

Embioptera.

Web-spinners in both the immature and adult stage feed on plant material, though it is reported that adult male Embioptera do not feed [35]. They do retain the full complement of mouthparts, as with the male shown here (Fig 7B), and the mandibles are used for grasping the female during mating.

Megaloptera.

Megaloptera larvae are carnivorous predators, feeding on a range of small aquatic invertebrates. Adults are generally thought to not feed [36] however they may ingest small amounts of fluid, or fruit, at least in a laboratory setting [37]. In gut content analyses, no significant amounts of solid food have been detected in adult dobsonflies, and it is hypothesized that they may rely more so on fat reserves accumulated during the larval stage [36]. The female Corydalus included here possesses sclerotized, functional mouthparts (Fig 7C).

Strepsiptera.

Male Strepsiptera are known to not feed, however the Xenos peckii male shown here (Fig 7D) features a developed set of mandibles used for cutting open the puparium during eclosion [38]. The remaining mouthparts are highly reduced and non-functional for feeding.

Lepidoptera.

Larvae of Bombyx mori, the domesticated silkworm adult included here, are known to feed continuously with a strong preference for white mulberry. Adult Bombyx mori do not feed and possess highly reduced, non-functional mouthparts.

Diptera.

The adult Ogcodes acrocerid spider fly shown here possesses highly reduced mouthparts (Fig 7E). Acrocerids are also known as small-headed flies; adults in the genus Ogcodes have relatively short lifespans, lasting from only 3 days to up to 4 weeks, and do not feed [39]. Some acrocerid genera have long proboscides used for feeding on nectar in flowers.

Ephemeroptera.

Mayflies have long been known to have a non-functional, inflated alimentary canal [15,16,18,28]. Combined with their exceedingly short adult lifespan and non-feeding behavior, mayflies serve as a useful exemplar in searching for other insects that may also possess a co-opted, air-filled gut.

As mentioned previously, the mechanisms for filling the inflated alimentary canal are unclear, even in mayflies, where the phenomenon has been known for some time. Connections to the tracheal system in Ephemeroptera have been proposed but not confirmed [18], and mapping of the mayfly tracheal system via micro-CT scanning indicates its isolation from the inflated gut [17]. It has been shown that rapid inflation occurs in mayfly females during oviposition, indicating that inhalation through the mouth provides air for inflation [18].

The mayfly subimago stage is unique among insects, and its developmental purpose remains unresolved (presumably a vestige of early hexapods like Zygentoma and Archaeognatha, which molt as adults) [40,41]. During this brief transitional stage of development, the insect is able to fly but is not yet sexually matured, and must molt again before becoming a reproductive adult. Subimago mayflies also do not feed, and the subimago Neocloeon triangulifer, micro-CT scanned as part of the previously mentioned respiratory morphology study ([17], and Herhold et al., in prep) displays a partially inflated alimentary canal (Fig 3). It is possible that reduction of digestive organs occurs in stages, whereupon the adult female is prepared to ingest air in response to oviposition. This does not resolve the question for males, however studies have suggested that ingestion of air may assist in ejaculation [18]. Pickles [15] suggested males are able to alter the degree of gut dilation, thereby changing their “specific gravity” to enable the rising and falling pattern of the mayfly “dancing flight”, however Harker [42] showed that mayflies with a constant gut volume were able to engage in swarming dance flight, indicating that rapid changes in gut air volume are not required for this behavior.

Strepsiptera.

All Strepsiptera males are short-lived, with a general life span of 3–6 hours, and must spend this time finding and mating with a receptive female [4]. Xenos peckii (Xenidae) males, for example, have been documented to live a maximum of four hours [38]; consequently, no adult males of Strepsiptera feed [3,4,20,43]. The females of Stylopidia (Strepsiptera excl. Mengenillidae), as neotenic and permanent endoparasites, have greatly reduced to vestigial mouthparts on the cephalothorax [4,44,45] and do not take in food as adults [6]. Rudimentary structures such as ommatidia, mandibles, maxilla, and labium can be seen on the female cephalothorax, but the region is primarily used for copulation and brood emergence [20,44]. The basal family Mengenillidae, sister group to all other extant taxa in the order, is the only family with free-living adult females that pupate outside of their hosts [4]. Female Mengenillidae are similar to other adult Holometabola as they do have mouthparts, however these females do not orally feed, also reliant on the host for nutrients [3,4,44].

The adult males of Strepsiptera (apart from the family Corioxenidae) generally retain moveable mandibles and maxillae [3,19,46]. The blade-shaped mandibles of Xenos peckii, the strepsipteran featured in this study, are used to cut open its puparium at an ecdysial suture line with a scissor-like motion, enabling the male’s emergence as an adult [38]. These blade-shaped mandibles are also present in males of the genera Elenchus (Elenchidae) and Stylops (Stylopidae) [3,4,19,47]. Use of the mandibles in breaking open the puparium has been documented for Stylops, but the same has not been confirmed by observation or recording for Elenchus [48].

In the family Corioxenidae, whose mandibles are vestigial to entirely reduced, the mode of emergence is likely a ptilinum [3] or a puparium cap that can be removed relatively easily due to a thin connection [20]. Emergence has been seen in male Elenchus tenuicornis via a ptilinum on the ventral side of the head between the eyes and below the antennae [49]. This differs from the positioning of the ptilinum in Diptera, but serves the same function. Kathirithamby [49] observed that male Elenchus tenuicornis undergo alternating inflation and deflation during the process of emergence, causing the ptilinum to repeatedly push against the pupal cap until it ruptures at a seam, very similar to the mechanism of eclosion used by teneral schizophoran flies to escape from the puparium. Though Elenchus tenuicornis exhibit this mode of emergence, they do still retain mandibles and maxillae [19,49]. The extent to which the ptilinum occurs in Strepsiptera is as yet unknown.

Regardless of variations in mouthpart structure and their use in eclosion, the presence of a large air space combined with adult non-feeding indicates co-opting of the alimentary canal in Xenos peckii and probably most or all Strepsiptera.

Plecoptera.

Studies on stonefly feeding have primarily focused on aquatic immatures, as these insects are essential to nutrient cycling in freshwater environments and serve as a useful bioindicator of water quality [50]. Anecdotal accounts led to the long-held assumption of widespread adult non-feeding in stoneflies [10], however this is not true for all taxa [32]. Isoperla (Perlodidae), a speciose Holarctic genus containing 313 species [51], has been found to feed on pollen, detritus, fungi, and algae in the imaginal stage [52,53]. The species involved in these gut studies, such as Isoperla nevada Aubert, 1952 or Isoperla grammatica Poda, 1761, were noted to have more developed mouthparts than other Isoperla [52,53]. Further, other Isoperla species from Great Britain and Sweden do not feed [10,54]. Thus, the feeding habits of this genus should be considered on a species-to-species basis. The volume of the air space in our female specimen (Fig 4) suggests a non-feeding adult life stage, although the presence of fully developed mouthparts is notable (Fig 7A).

Numerous species within the superfamily Perloidea feed during their adult stage [55], but the idea that adult stoneflies do not feed was perpetuated by the insistence that mouthparts are often reduced at the imaginal stage [10]. Although some Isoperlinae have somewhat reduced mouthparts, the presence of sclerotized mandibular teeth is incredibly common [32], and both adult feeding and non-feeding behaviors have been documented throughout Plecoptera [53,55–57].

As shown in Fig 4, the presence of a substantial number of eggs in the anterior portion of the stonefly abdomen may limit the size of the inflated alimentary canal. The weight of a female stonefly after egg deposition can drop by approximately 30% [58] as they take up a majority of space within an individual’s abdomen. Stoneflies generally have smaller clutch sizes than mayflies, thus depositing fewer eggs, but systellognathan stonefly eggs have a hard exterior chorion [33,34] thought to protect against environmental destruction before nymphal development (note more dense, sclerotized shells in posterior eggs; Fig 4) [59]. We hypothesize that this could limit the amount of available air space in the gut.

Embioptera.

Female adult webspinners differ from males in that the females have a large crop, stores of fat, and a proventriculus that aids in digestion of food [60]. Adult male webspinners lack these structures and tend to not feed after their final molt [35]. Adult male Metoligotoma (Australembiidae) are one of the few Embioptera that have been recorded to feed with their robust mandibles and enlarged palpi [61]. This suggests that they live longer than other male embiids, which typically die quickly after mating [62].

Oligotoma nigra nymphs have well-developed maxillary palps and chitinized mandibles with many denticles, indicating that they engage in biting and chewing feeding habits [63]. The mouthparts of the adult Oligotoma male retain much of their structure and utility, likely because webspinner males use them to grasp females during mating [35]. The thinner abdomen, as well as simpler mandibles, are both indications that adult male Oligotoma do not feed [63].

The inflated air space occupies the entire length of the body (Fig 2B). This non-feeding adult male specimen serves as an excellent example of a likely co-opted alimentary canal.

Megaloptera.

Immature life spans of the predaceous aquatic stage of megalopterans are well-known to be quite long and merovoltine, sometimes taking years to reach maturity [64]. In contrast, less is known about the reproductive, terrestrial life history of Megaloptera as the adult life spans are limited, usually only lasting between one week to one month [65,66]. It was previously thought that adult megalopterans generally do not feed, however several studies have indicated that adult females may feed on pollen, detritus, and male spermatophores to obtain substantial energy, although it is still widely accepted the females largely depend on larval energy reserves [36]. Males of Corydalus are unable to feed on solid food due to their large mandibles but have been observed licking the juice off of fruits [37] or honey [67] in laboratory settings, a behavior also observed in neotropical Megaloptera [36]. Adult megalopterans have been found to visit flowers for nectar or pollen [68–70], feed on tree sap [71], and consume small insects [72] in nature. As there has been much debate on whether all species of adult megalopterans feed, it is important to study the gut anatomy amongst genera and species.

Lepidoptera.

Across the approximately 180,000 species of the order Lepidoptera, there is considerable variation in feeding behaviors and mouthpart development. Many moth species do not feed as adults, while adult butterflies often use proboscides in nectivorous feeding [73,74]. Most notably, adults of all species in the silk moth family Saturniidae have reduced mouthparts and live abbreviated lifespans from one to two weeks, with an emphasis on reproduction and dispersal [74].

Interestingly, adults that do not feed and have reduced or atrophied mouthparts also occur in groups of Lepidoptera that are well known known to feed, including some hawk moths (Sphingidae) and ghost moths (Hepialidae). Hawk moths have a range of mouthpart development, including long proboscides for accumulating nectar [75] and host plant pollination. However, a recent study has shown that several species have lost the adult ability to feed and have reduced or nonfunctional proboscides [9].

As a model organism, the domestic silkworm Bombyx mori has been well known for its reduced mouthparts and non-feeding adult. The inflated alimentary space shown in the scanned adult occupies only the anterior portion of the abdomen, however it fills a substantial portion of the body cavity laterally and dorso-ventrally. In conjunction with the reduced mouthparts and known non-feeding behavior, it is likely this specimen also demonstrates a co-opted alimentary canal.

Diptera.

Along with Lepidoptera, Diptera is one of the four major holometabolous insect orders that, along with Hymenoptera and Coleoptera, comprise over 80% of all insect species. The approximately 170,000 known species of flies feature an extremely wide array of life histories and habitats [76]. Typical of the order Diptera, mandibles are generally lost (though retained as fine lancets or thin blades in the females of some blood-feeding groups); labial palps and glossae are fused into a pair of fleshy lobes that sponge up liquid food (in Brachycera these lobes have pseudotracheae, fine chinized tubes which assist in liquid feeding) [77–80]. Adult non-feeding is known in several groups, including several species of non-biting midges (Diptera: Chironomidae). For example, Belgica antarctica, a flightless midge and one of the only completely terrestrial animals native to Antarctica, spends most of its two-year lifespan in larval stages, followed by an abbreviated non-feeding adult stage.

Acroceridae is an ancient, phylogenetically isolated, small family of stout-bodied Brachycera whose larvae are parasitoids of spiders. There are dramatic differences in mouthpart development among genera, varying from highly vestigial and functionless, as in Ogcodes, to genera like Eulonchus and Lasia, which have extremely long proboscides used to feed on nectar and pollen from deep flowers [81,82]. Refer to S1 File for detailed descriptions of mouthpart morphology and characters.

Ogcodes flies are reported to not feed as adults [39]. The specimen included here features the most extreme example of an essentially completely air-filled abdomen, which appears to be divided between the crop and the alimentary canal (Figs 2E, 4). It is notable that the alimentary air space is restricted to the abdomen, possibly due to the lack of available space in the thorax, dominated by large flight muscles, and the head, which is limited in size overall.