Transformation of internal head structures during the metamorphosis of Chrysopa pallens (Neuroptera: Chrysopidae)


 Background The metamorphosis is a complicated but very interesting process because of the highly dynamic transformation in sheath. Very few studies had coverage on the head muscles of larvae, pupae, and adults. Most of these studies were focusing on the model organisms about the rough changes of the external and internal tissues or the time of metamorphosis based on the traditional methods. In our study,the skeleto-muscular system of head, as well as the brain of Chrysopa pallens (Rambur, 1838) from larvae to adults are described in detail for the first time by the technology of micro computed tomography (µ-CT). The transformations of these systems during pupal stage are studied for the first time.Results The morphological differences and functional adaptations between the stages are assessed. Muscles are distinctly slender in larvae than in adults with a significantly larger quantity. A larger brain with improved sensory perception is suggested to be essential for dispersal, mating and flying for adults. For the pupae, the results show that the histolysis of the muscles happens in first third of the pupal period and their reconstruction happens in the following days. The brain exists all along.Conclusion We suggest the transformations of the skeleton occur earlier than the musculature. Most of the transformations are related to tasks they play in the developmental stages.


Background
Holometabola ( = Endopterygota) with approximately 800,000 described species comprise about two thirds of the known animals [1]. Driven by different factors, the outbursts of diversifications took place in different megadiverse subgroups, for example, the co-evolution between flight apparatus and angiosperms [1,2,3,4]. Nevertheless, the metamorphosis from larvae to pupae and from pupae to adults might be a crucial feature for the evolution of the corresponding lineage [5]. This includes the ontogenetic developments including diet, reduced intraspecific competition between juveniles and adults, et al. [1].
Most studies of the metamorphosis of holometabolous insects were focusing on the model organisms, like the fruit fly Drosophila melanogaster. Some of these studies were about the rough changes of the external and internal tissues or the time of metamorphosis based on the traditional methods [6,7], some were about the genes expression during metamorphosis based on genome approach [8,9]. Also, the nerves of the mealworm Tenebrio molitor during metamorphosis [10,11] and the transformation of the abdominal muscles of the blowflies during metamorphosis (Calliphora [12]) were focused. However, Oertel [13] omitted the detailed information of the cephalic musculature of honeybee Apis mellifera. Polilov & Beutel [14,15] focused on the effects of miniaturization and phylogeny by comparing different beetles. Ge et al. [16] omitted the transformations during pupal stage of Chrysomelinae beetles. Even though there have been several cases of studies on head musculature of different species of Neuroptera reported, such as Osmylus fulvicephalus [17], Coniopteryx pygmaea [18], and Sisyra terminalis [19], Nevrorthus apatelios [20], very few detailed studies have been carried out on the metamorphosis of Neuroptera. Occasionally, these skeleton-muscular features have been used in Phylogenetic analyses [20,21,22,23,24]. Almost the same happened to Megaloptera and Raphidioptera, which belong to Neuropterida. The head muscles of Chauliodes formosanus and Sialis flavilatera of Megaloptera [25,26] and Raphidia flavipes of Raphidioptera [27] were described in detail based on the traditional methods. Recently, the larval head muscles of Raphidia (Phlaeostigma) notata were described by Beutel & Ge [28] based on the 3D reconstruction method. In most cases, the studies of the cephalic nervous system were included in the studies of the head musculature of larvae or adults. The nervous system of the pupae was rarely studied. Recently, the 4 th day pupae were reconstructed in detail by Ge et al. [16]. The metamorphosis is a complicated but very interesting process because of the highly dynamic transformation in sheath. Very few studies had coverage on the head muscles of larvae, pupae, and adults. Therefore, the morphological transformations during metamorphosis are presently still very insufficiently known.
Finally, by concerning the recent studies about the morphological methods [29,30,31], the nondestructive method-computed tomography (μ-CT) and the conspicuous lack of information on metamorphosis of lacewings induced us to execute this comparative study of the head structures. The focus of this study was the detailed documentation of transformations in the head muscular system, the cephalic nervous system between different developmental stages of the green lacewings Chrysopa pallens. Chrysopa pallens (Rambur, 1838) belonging to Chrysopidae. The green lacewing is one of the most common encountered families of insect of the order Neuroptera. They distributed in all major biogeographic regions of the world [32,33]. They are used as predacious biological control agents of insect pests such as aphids in many agricultural applications [34,35,36,37]. Within this family, members of genus Chrysopa and Chrysoperla, for instance, Chrysopa pallens (Rambur, 1838), and Chrysoperla carnea (Stephens), have been mass reared and sold by numerous commercial insectaries.
The adult cephalic and thoracic musculature of another species of Chrysopa, Chrysopa Plorabunda, has already examined in detail by Miller [38]. However, the study was based on traditional methods and the homology of the muscles remained ambiguous. Some muscles were apparently overlooked such as tentoriomandibularis muscles [38]. Some muscles were subsumed under one name whereas they were treated as separate units by Wipfler et al. [39]. In the present study, we documented the muscles of the head, the cephalic nervous system from larvae to adults, by micro-CT reconstruction techniques, with the complete data of pupal stage included.

Examined specimens
The Chrysopa pallens (Rambur, 1838) were raised in the laboratory with temperature 25℃ and humidity 75%. Three samples were collected every day from the first day of pupae to the emergence.
All materials were preserved in 75% ethanol for less than 24 hours before dehydrated. X-ray computer tomography All materials used for X-ray micro-computed tomography (µ-CT) were dehydrated in pure n-propanol, then in ethanol solutions from 75% to 100% stepwise. Then they were dried at the critical point (Leica EM CPD 300). The specimens were scanned by an X-radia Micro CT-520 scanner at Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences (beam strength: 40KV, absorption contrast) and X-radia Micro CT-400 scanner at the Institute of Zoology, Chinese Academy of Sciences (beam strength: 60KV, absorption contrast).

Three-dimensional reconstruction (3D)
The muscles and the brains of the larvae, pupae and adults were reconstructed and smooth with

Terminology
The terms used for the head muscles followed the terminology of Wipfler et al. [39].

Results
The head skeleto-muscular system and the cephalic nervous system of the third instar larvae, the pupae (from Day 1 to Day 12), and the adults of Chrysopa pallens (Rambur, 1838) are described.

Skeleto-muscular system
The external structures of head are described. During the pupal stage, from Day 1 to Day 4, the skeletal system is almost same to what is in the 3 rd instar larvae, thus only the latter is described in detail. In Day 5, the larval cuticle cracked, and the newly present structures keep themselves in the following 7 days, so only the pupae of the 11 th day, which is well developed, is described in detail.
Muscles in pupal stage are described in this section, too. The description of head muscles is in tables 1-3.

General appearance
Third instar larvae (Figs 1A,2). Body of mature, living third instar larva fusiform and humped. Length 7.00 mm and height ~1.30 mm. Cuticle light brown with dark brown markings dorsolaterally.
Spinules and long microsetae present dorsally. All setae smooth, dark brown to light brown. Flat head 1.00 mm in length and 0.70 mm in width. Thorax unsclerotized with rows of short, acute setae. Legs slender and well developed, inserted on semimembranous ventrolateral articulatory areas posteriorly.
Lateral tubercles broadly cylindrical dorsolaterally and tapering distally with elongated setae. Long setae all tapering and hooking at tips. Tubercles and long setae carry the debris for camouflage.
Pupae from Day 1 to Day 4 (Fig 3). Pupae immobile adecticous exarate type. Cuticle light brownyellow. Body C-shape with 0.50 mm in length and 0.30 mm in width. Head bends inward, morphologically almost same to larvae. Segments of thorax and abdomen similar in shape. Lateral tubercles smaller and long setae disappeared. Cocoon 0.40 mm in length and 0.30 mm in width, with dead aphids covering on cocoon (Fig 1D).
Pupae from Day 5 to Day 10 (Figs 3,4). Color and body shape stay same. In Day 5, larval cuticle cracked and wings present. Larval cuticle gathers under abdomen in cocoon ( Fig 1E). Pharate adult 6.00 mm in length and 2.50 mm in height. Head 0.15 mm wide and 0.12 mm long. Compound eyes, basal antenna, labrum, and mandible similar to adults. Color of labrum and mandibles turn red to crimson from Day 6 to Day 10. Compound eyes red to metallic black-red. Maxillary palps, labial palps, and curly antenna present with milky color. Frontoclypeal sulcus present. Wings become larger in size. Prothorax, mesothorax, metathorax, and legs similar to adults in shape. Short setae present on frons.
Pupae Day 11 (Figs 1B,3,4). Head hypognathous, nearly triangular in frontal view, yellow to pale brown from vertex to mouthparts. Posterior vertex slightly concave. Compound eyes hemispherical, metallic black, occupying half of head width. Ocellus absent. Antennas locate between compound eyes. Antennomeres extremely elongated, covering on sides of body. Clypeus broad. An indistinct suture present between clypeus and labrum. Lateral gena strongly round. Ventrally, labium connects with maxilla.
Adults (Figs 1C,5). Same shape and color to Day 11 pupae. Posterior vertex concave. Compound eyes large and metallic black, composed by numerous small and hexagonal ommatidia. Ocellus absent.
Scapus swollen in antennal socket. Antenna filiform and almost as long as body length. Head nearly wedge-shaped in lateral view, gradually narrowing to mouthparts. Ecdysial line vestigial.
Frontoclypeal sulcus and frontogenal suture present. Dorsolateral longitudinal furrow extends from dorsolateral margin of hind head capsule to mandible articulation. Lateral occipital lobes slightly exposed and hemispherical. Frontogenal suture connects anterior antennal fossa with dark anterior tentorial pits. Subgenal suture above mandible articulation vestigial. Lateral clypeus round. Anterior clypeus concave slightly with convex median line.
Pupae from Day 3 to Day 10 (Fig 7). From Day 3, new tentorium present, including two separated arms. Boundary of ata and pta indistinct before Day 6. In Day 10, tb present.

Antenna.
Third instar larvae (Fig 2). Antenna glabrous and multisegmented in a slightly elevated socket. Basal

Mandible.
Third instar larvae (Fig 2). Mandibles strongly elongate, slender with apical parts, slightly upturned, longer than labial palps, closely connected with elongate maxilla. Sucking channel enclosed by More and more muscle fibers and bundles present in following days. In Day 12, almost all muscles present in bundle form.

Cephalic nervous system
The main elements of the central nervous system are brain and subesophageal ganglion. The latter is the first ganglion of ventral nerve cord. The two with the frontal ganglion are the main elements of the cephalic nervous system. Cerebrum, suboesophageal complex, and frontal ganglion Third instar larvae (Fig 13-larvae). Size of brain and suboesophageal ganglion (sog) about 20% that of entire head capsule. Brain composed by protocerebrum, deutocerebrum, and tritocerebrum.
Protocerebrum dumbbell-shaped and optical nerves extremely slender with very slightly round lobe.
Two thin antennal nerves originate from slightly protruding region of deutocerebrum. Frontal connectives originate from tritocerebrum and circumoesophageal connectives continuous with tritocerebrum. Sog ovoid-shaped below tb. All slender nerves of labium, maxilla, and mandible originate from sog. Frontal ganglion triangular, connecting with the protocerebrum and tritocerebrum by three curved frontal connectives.
Pupae Day 11 (Fig 13-pupae). Volume of brain and suboesophageal complex small, occupying about 12.5% that of head capsule. Protocerebrum unrepresentative dumbbell-shaped. Optical nerves cylindrical with slightly round lobe. Antennal nervus slender and bending upwards. Tritocerebrum bears circumoesophageal connectives. Suboesophageal complex nearly oval. Front ganglion triangular and connected by two curved frontal connectives.
Adults (Fig 13-adults). Volume of brain and suboesophageal complex occupies about 33.3% that of head capsule. Protocerebrum dumbbell-shaped with two large optic neuropils. Suboesophageal complex oval. Triangular frontal ganglion connected by three nerves like larvae.

Pupal brains
Transformation of brains from Day 1 to Day 11 is illustrated in Figs 10,11,14. In Day 1, brain becomes small and simple. Antennal nerves, optical neuropils, and mouthparts nerves strongly short.
Frontal ganglion disintegrated. In Day 2, brain strongly compressed and suboesophageal ganglion separated from brain due to disappearence of circumoesophageal connectives. From Day 3, brain stops compression but becomes more and more larger in following days. In Day 9, slender antennal nerves present. In Day 11, frontal ganglion present.

Functional adaptations in larvae and adults
The morphology of the larvae and adults differ significantly. Nearly all character systems would be affected during the process of metamorphosis. The homologization of the muscles between all stages is concluded in tables 4. Tables 5 show the homological patterns of muscles reported previously.
The specialization of larvae and adults, which could lead to the possibility of living in different habitats and feeding on various food substrates, results in a reduced intraspecific competition between the stages. This was addressed as one factor contributing to the unparalleled evolutionary success of Holometabola [1,4]. It is also conceivable that a division of ecological selection between developmental stages may bring about the selections for reduced equipment in larvae, which do not need elaborate sense organs like the sensillum on antennae and the compound eyes of adults.
Besides, the organs for dispersal over long distances are absent in larva. The main target for larvae is to be feed and accumulate energy-rich substances in fat body. Whereas, the principal functions of the adults are dispersal and reproduction. The present study reveals how these divergent functions affect the metamorphosis of different structural elements.
The most conspicuous change is the orientation of the head: it is prognathous in larvae but orthognathous in adults. Functionally, this may relate to the height of the body. The larval legs are relatively short. Thus, the body would almost touch the ground and the preys would be almost as tall as the larvae. The prognathous head can help the sucking channel piercing the upper part of the prey.
However, the adult's legs are longer than larval legs and the mouthparts are relatively higher than larvae. The orthognathous mouthparts are more conducive for capturing and getting the small preys.
The downward orientation of the mouthparts from larvae to adults also closely relates to the head morphology between different stages, such as the concave submentum, the broader vertex of the adults. Additionally, the wedge-shaped capsule of the adult is in favor of the development of the mouthparts and the cephalic nervous system, as well as the development of the strong muscles, such as 0an1 and 0an2. For most holometabolous insects, the upward or downward orientation from larvae to adults may possibly depend on their styles of mouthparts in larvae, such as the upward orientation in Coleoptera [16].
The necessity to find a potential mating partner requires a far more complicated sensory system in the adults than is present in larvae: instead of simple stemmata, the complex compound eyes are present. This change in the visual system also requires strong modifications in the brain, notably in the optic lobes which greatly increase in size. Similarly, the antennae are greatly elongated in adults.
The antennal nerves also become larger in size than previous stages. To ensure controlling the movements of adult antennae, a more complex muscle system is required. Three extrinsic and four intrinsic muscles are present in antennae of adults, whereas only four small extrinsic muscles and no intrinsic muscle present in that of larvae. It is conceivable that the intrinsic muscles could have a more effective control towards the flight mechanism than the extrinsic muscles. Additionally, from larvae to adults, the segment number of the labial palp is the same but two more intrinsic muscles and one more extrinsic muscle are presented in adults. The Maxillary palps is absent in larvae, but the five-segment palp, four more intrinsic, and two more extrinsic muscles are presented in adults.
In addition to the modifications mentioned above, a muscle intrinsic muscle of maxillary stylet (imms) connecting the dorsal wall and ventral wall of stylet is presented in larvae but absent in adults. The muscle is also reported in the larvae of Nevrorthus [40]. Functionally, it probably controls the movement of stylet. The volume of the sucking channel is springy thanks to the muscular contraction, which assists in sucking the fluids.
In the pharynx musculature, ten muscle bundles are presented in larvae and nine are presented in adults. The only one missing is the muscle M. prelabiohypopharyngeal muscle (prhy) which has unclear homology. It is closely associated with the solid food that the adults feeding on. The similar muscle is also presented in the larvae of Nevrorthus [40]. Functionally, it might have something to do with stabilizing the structure of labium and anterior pharynx in larvae. However, this muscle may limit the movement of labium in adults. It is reasonable that the muscle prhy is absent in adults, whose labium needs a more flexible movement.

Transformation in Pupae
In general, the mature pupae resemble the adults in almost all skeletal elements except for the absence of the dta and the curly elongated antenna. Aside from these skeletal features, there is another major transformation taking place in the orientation of the head. The larvae are clearly prognathous with an angle of approximately 200° between longitudinal body axis and longitudinal axis of the mouthparts (Fig 15). The adults are orthognathous with an angle of 135°. From the Figure   3, we found that the pupae have the angle 60° from Day 1 to Day 4 but have the angle 90° from Day 5 to Day 11. Comparing these angles from larvae to adults, it is easy to assume that the orientation of the mouthparts is a sudden shift rather than a continuous shift. The sudden shift from 200° to 60°m ay take place during pupating and with the similar angle in the following four days. After the larval cuticle being cracked in Day 5, the angle is 90° and become a little bigger after the cocoon breaks.
The angle becomes 135° once the emergence happens. It was suggested in Ge et al. [16] that "anterior orientation of the mouthparts is a continuous shift rather than a sudden reorientation and takes place more or less continuously during the six days of pupal metamorphosis." Considering their data only contains one day as the sample of pupae stage, it is hard to compare if the two studies are talking about the one or two phenomena.
Based on the results, we found that the construction of the new cuticle and the histolysis of the internal structures (such as muscles and tentorium) almost happen in the first third of the period. At the beginning of the pupal stage, the muscle fibers can be recognized easily but smaller than larvae.
With the presence of the new cuticle and the histolysis of the larval muscles, some new muscle granules are presented in Day 4. However, the great increase of the muscle granules happens in Day 6. We guess the most muscle bundles would take shape in the next day. In fact, the data of Day 7 prove it. This is consistent with the study of honeybee whose muscle bundles are presented after 150 hours of pupation [13]. However, the musculature of the mature pupae appears frayed or do not attach to the skeletons, such as M. craniomandibularis internus (0md1), P6 and P7 that only one end attached to the sclerites. This phenomenon also exists in Coleoptera [16].
We also found that the modifications of skeleton happen earlier than the internal soft parts during metamorphosis. Even though the compound eyes are already presented in the pharate adults, the optic lobes of the brain are still undeveloped in the last day. In addition, the results show that the brain and the suboesophageal ganglion are not disintegrated during the pupal stage. Indirectly, the importance of central nervous system in development and metamorphosis during the life history is verified. Concerning the beginning time of the modification, the musculature lags behind the nervous system. We thus conclude that in Chrysopa, themodifications of the skeleton begins earlier then the nervous system and the musculature. All these systems would develop well in the end of pupal stage or after the emergence. It is consistent with the research of Oertel [13] about the transformation of the honeybee, as well as the study of leaf beetle [16]. However, due to the diversity of events occurring throughout the Holometabola, the observations in only several species is insufficient and certainly this interpretation is preliminary.

Conclusions
Our study showed that muscles are distinctly slender in larvae than in adults with a significantly larger quantity. Most of these transformations are related to tasks they play in the developmental stages. A larger brain with improved sensory perception is suggested to be essential for dispersal, mating and flying for adults. For the pupae, the results showed that the histolysis of the muscles happens in first third of the pupal period and their reconstruction happens in the following days. The brain exists all along. Insect metamorphosis is arguably among the most complex processes in animal life. It almost covers the knowledge from morphology, neurology, and developmental biology [7,9,41,42,43,44]. It is apparent that more detailed comparative studies involving representatives of all principal endopterygote groups are required. Future studies involving broader taxon sampling with the advanced methods, may lead to a better understanding of this remarkable phenomenon, which apparently played an important role in insect evolution [4].     "+"= present""= absent