# Morphology of the Preimaginal Stages ofLasioptera donacis Coutin (Diptera: Cecidomyiidae), a Candidate Biocontrol Agent for Giant Arundo Cane

Authors: Donald B. Thomas; John A. Goolsby
Journal: Psyche
 (2015)

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## Abstract

The larval stages ofLasioptera donacis Coutin consist of three instars which develop within the mesophyll of the leaf sheaths of Arundo donax (L.) (Poaceae). The larvae feed 	aggregatively on mycelia of an ambrosia fungus. The third instars are similar to other members of the genus except for a three-pronged spatula (typically two-pronged) and five lateral papillae (typically four) and with a nonbristled first instar. A related species, L. arundinis (Schiner) which breeds on fungus in Phragmites (Poaceae), also has a three-pronged spatula and five lateral papillae but has a bristled first instar. The third instar of L. donacis has a feeding and a nonfeeding prepupal stage. Papillae associated with the spatula are sensory organs, sensilla chaetica, sensilla trichodea, and sensilla ampullacea, perhaps related to extraoral digestion of the fungal mycelia. Pupation occurs in the host plant within a silken cocoon. Egression of the adult is through an escape hatch excavated by the third instar.


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## Body

## 1. Introduction

The genusLasioptera Meigen 1818 is defined taxonomically by structures on the postabdomen and ovipositor of the adult female [1]. These structures are mycangia, organs which function in the transport of fungal conidia. The gall midges of this genus are obligate symbionts of a fungus on which the larval stages feed [2, 3]. Females collect the conidia and oviposit them with the eggs into the host plant. The fungus is an opportunistic endophyte capable of exploiting living plant hosts. The fungus rewards its symbiont for providing transport to potential host plants by providing nourishment in the form of an ambrosia, a filamentous mycelial mass which fills the tunnels inhabited by the larvae [4]. In these cases of symbiosis the inoculation of the fungus into the plant by the insect has been documented for the Platypodidae (Coleoptera) [5], the Siricidae (Hymenoptera) [6], and the Cecidomyiidae (Diptera) [3, 7]. Coutin and Faivre-Amiot [8] first discovered and outlined the tritrophic relationships among this insect,Lasioptera donacis Coutin 2001, the fungus, and its host plantArundo donax L. (Poaceae: Poales). These authors also described and illustrated the ovipositor with its mycangia on the adult female. The preimaginal stages have morphological features which adapt them to this resource niche and the objective of this study is to describe some of those adaptations.General treatises on the morphology of larval cecidomyiids have been published by Otter [9], Möhn [10], Gagné [11–13], and Mamaev and Krivosheina [14]. The larval stages of the two most economically important cecidomyiids, the Hessian fly,Mayetiola destructor(Say 1817), and the sorghum midge,Contarinia sorghicola (Coquillett 1899), have been described in considerable detail primarily in the context of the damage to the host plant [15, 16]. Illustrations and descriptions of larval Lasiopteridi include Gagné et al. [17, 18] and Boe and Gagné [19]. The larvae of 20 species ofLasioptera were described in a taxonomic study by Möhn [10] which included the descriptions of new species based on larval specimens.With few exceptions the larvae ofLasioptera spp. are narrowly host plant specific. BecauseArundo donax (L.), the giant reed or carrizo cane, is an invasive plant that has spread into the riparian systems of the southern United States [20–22],L. donacis is under consideration as a potential biological control agent [23]. In that context, the basic biology of this insect is of some importance and the morphological adaptations of the immature stages indicate the specific means by which it exploits the fungus within its host plant. A related fungus feeding species,Lasioptera arundinis (Schiner 1854), breeds in a related host, the common reed,Phragmites australis (Cav.) Trin. (Poaceae), and hence has a life cycle and morphology similar to that ofL. donacis. Thus, a secondary objective of our study was to ascertain the differences in larval morphology betweenL. donacis as they appear in the original description by Coutin [24] and observed by us, with those ofL. arundinisas described by Möhn [10] and Rohfritsch [4].


## 2. Materials and Methods

### 2.1. Rearing ofLasioptera donacis

The late instars and pupae examined in this study were dissected from host plant material collected near Montpellier, France (43°40′N; 04°01′E), and Vandellos, Spain (41°01′N; 0°50′E). These were shipped by air courier to the USDA-APHIS Arthropod quarantine facility near Edinburg, TX, where a research colony is maintained. Eggs and first instar specimens were collected by inducing oviposition by colony adults placed in cages with potted Arundo plants in a greenhouse with temperatures maintained at 23° ± 3°C. Similarly, RH was targeted to 100% but actual recorded RH varied around a mean of 80%. Light cycle was natural. According to Coutin and Faivre-Amiot [8]Lasioptera females seek preexisting holes in the leaf-sheath, made by a chloropid, for oviposition. Thus, in order to rearL. donacis we artificially perforated the leaf sheathes of potted plants with a fine pair of forceps. In our laboratory colonies all egg masses found were in the mesophyll channels underlying these artificial perforations. The colony at Edinburg was first established in 2007 and continues to the present day.


### 2.2. Preparation of Specimens

For chaetotaxy some larval specimens were cleared and mounted following the KOH-Clove oil method of Gagné [12] while others were cleared with d-limonene (Histoclear, National Diagnostics, Atlanta GA). Some of the cleared specimens were stained with aceto-orcein or eosin to enhance visualization. Measurements of larvae and larval structures were made on a Leica MZ16 dissecting microscope with Leica application suite software. Photomicrographs of third instars were made on a Nikon Eclipse E400 microscope. For first and second instars whole specimens were mounted on slides using Permount (Fisher Scientific, Hampton NH) and photographed with a Nikon SMZ645 microscope. For internal organs larvae were mounted in paraffin, sectioned with a rotary microtome, and stained with methylene blue or eosin. Chaetotaxy follows Gagné [12] and Möhn [10]. Nomenclature for the sensory organs is that of Zacharuk and Shields [25].


## 2.1. Rearing ofLasioptera donacis

The late instars and pupae examined in this study were dissected from host plant material collected near Montpellier, France (43°40′N; 04°01′E), and Vandellos, Spain (41°01′N; 0°50′E). These were shipped by air courier to the USDA-APHIS Arthropod quarantine facility near Edinburg, TX, where a research colony is maintained. Eggs and first instar specimens were collected by inducing oviposition by colony adults placed in cages with potted Arundo plants in a greenhouse with temperatures maintained at 23° ± 3°C. Similarly, RH was targeted to 100% but actual recorded RH varied around a mean of 80%. Light cycle was natural. According to Coutin and Faivre-Amiot [8]Lasioptera females seek preexisting holes in the leaf-sheath, made by a chloropid, for oviposition. Thus, in order to rearL. donacis we artificially perforated the leaf sheathes of potted plants with a fine pair of forceps. In our laboratory colonies all egg masses found were in the mesophyll channels underlying these artificial perforations. The colony at Edinburg was first established in 2007 and continues to the present day.


## 2.2. Preparation of Specimens

For chaetotaxy some larval specimens were cleared and mounted following the KOH-Clove oil method of Gagné [12] while others were cleared with d-limonene (Histoclear, National Diagnostics, Atlanta GA). Some of the cleared specimens were stained with aceto-orcein or eosin to enhance visualization. Measurements of larvae and larval structures were made on a Leica MZ16 dissecting microscope with Leica application suite software. Photomicrographs of third instars were made on a Nikon Eclipse E400 microscope. For first and second instars whole specimens were mounted on slides using Permount (Fisher Scientific, Hampton NH) and photographed with a Nikon SMZ645 microscope. For internal organs larvae were mounted in paraffin, sectioned with a rotary microtome, and stained with methylene blue or eosin. Chaetotaxy follows Gagné [12] and Möhn [10]. Nomenclature for the sensory organs is that of Zacharuk and Shields [25].


## 3. Results

Lasioptera donacis has three larval instars that are passed entirely within the spongy mesophyll layer of the stem leaf sheaths ofArundo donax. The larvae are essentially colonial, feeding in clusters of about the same stage of development, and presumably from the same clutch of eggs, but at times in larger aggregations that can include two instars likely from successive ovipositions. Hodin and Riddiford [26] demonstrated that the nutritional content of the mycelium was a factor in the inducement of paedogenesis in fungus feeding midge larvae. Characterized by a fourth instar that lacks the spatula, paedogenesis has not been reported inLasioptera, nor did we observe this phenomenon inL. donacis.Egg masses with fungal conidia were found in the medullary channels soon after exposure of the perforated plants to female midges. The first instars are free-living for at least several days after hatch because it typically requires about a week after inoculation for the dark mass of fungal mycelia to grow and accumulate (Figure1). Invariably the second and early third instar larvae were found in channels of the medullary tissue, typically with the head end immersed in, and presumably feeding on, the dark yeasty mass of mycelia (Figure 2). The third instar has a feeding and a prepupal nonfeeding stage. At the end of the feeding stage the third instar spins a silken cocoon in which it will eventually pupate, still occupying the fungus filled channels of the leaf sheath mesophyll. The nonfeeding stage is found in the cocoon which is open at one end. Just prior to spinning the cocoon the larva cuts an escape hatch in the leaf wall from which the imago will eventually egress. It uses a sclerotized armature on the prothorax, common to almost all cecidomyiids, the “sternal spatula,” to excavate the emergence hole [26]. However, this hole does not open freely to the exterior; rather, a thin flap of epidermal plant tissue seals the hole. The larva then closes the cocoon by spinning a disc shaped, silken operculum. When the adult emerges from the escape hatch it simultaneously ecdyses from the pupal cuticle, characteristically leaving the pupal exuvium protruding from the exit hole in the leaf sheath (Figure 3).Figure 1
First instars with a mass of black yeasty mycelium (center) at approximately ten days after oviposition. White hyphae are extending out from the old egg clutch into the plant tissues.Figure 2
Third instars are found with the ambrosia fungus, a brown yeasty material, in the mesophyll layer ofArundo donax.Note the silken cocoons at center and upper left.Figure 3
Pupal exuviae protruding from exit holes in the leaf sheath following emergence of the adults.Egg: typically dipteran in configuration: elongate, length about 0.5 mm, blunt at one end, tapering to a point at micropylar end. No respiratory horns visible. Color pinkish transparent. Observed in masses of 15 to 25 (Figure4). Under high magnification one can see within each egg yellowish vacuoles that are presumably yolk protein.Figure 4
A clutch of eggs in a mesophyll cell ofArundo donax.First instar: color pale sometimes pinkish, translucent, elongate fusiform in body shape; length 0.4–0.5 mm at eclosion. Two conspicuous orangish spots visible in abdomen at eclosion (Figure5). These conspicuous spots may be the same vacuoles of yolk protein visible in the eggs. Soon after eclosion the material diffuses through the gut tract as the larva crawls away from the egg mass. Bolwig’s organ, the subcuticular eyespots, is reddish and found between head capsule and prothorax (Figure 6). The cuticle is transparent and the head capsule, tentorium, and spiracles are nonsclerotized. The head capsule at this instar is almost as wide as the body segments. The antennae are orbicular with terminal nipple. The microverrucae of the cuticle are spinulate, particularly in the rows at the anterior margins of the body segments.Figure 5
A recently hatched first instar. The orange spots are vacuoles of yolk carried over from the egg.Figure 6
First instar, head and thoracic segments. The ovoid, red objects between the tentorial arms are Bolwig’s organs. Note the rows of cuticular denticles.The first instar has a complement of haired papillae arrayed in number and position characteristic for the Lasiopteridi [1]: six dorsals on each thoracic segment and first eight abdominal segments; a pair of pleural papillae on each side; and a pair of hind-ventrals on all postcephalic segments except penultimate and terminal segments. Penultimate segment has four ventrals and terminal segment with eight terminal haired papillae, the hairs 3-4 μm long, about twice as long as papillus on which they are seated. The eighth-abdominal segment has larger pleural papillae, the hairs of which are 4.5–5.3 μm long (Figure 7). The first instar does not have the sternal spatula nor does it have discernible sternal or lateral papillae. The body (Figure 8) lacks the long (30 μm) bristles and numerous spines reported inL. arundinis [27].Figure 7
First instar, terminal abdominal segments showing haired papillae, the longest of which are those attendant to the spiracles.Figure 8
The first instar does not have long mycangial bristles as reported inLasioptera arundinis. Note that the head capsule is nearly as wide as the prothorax.Second instar: color pale yellowish, length 1.0–1.75 mm (n=4). Second instar larvae do not have the sternal spatula. Head capsule distinctly smaller in proportion to the body segments compared to the first instar and with antennae more elongate (Figure 9). The cuticle is transparent and with backlighting under the compound microscope in each abdominal segment fat body is visible as three large darkened amorphous masses of tissue: one at middle and one on each side. Lateral papillae on thorax have 11–15 μm long bristles while those on eighth-abdominal segment are 16–19 μm long. Spiracles are present on prothorax and first eight abdominal segments.Figure 9
The head, neck, and prothorax (with spiracles) of the second instar. The spatula is absent. The dorsal and pleural papillae are setiform. The sternal and lateral papillae are absent.Third instar: color orange in life fading to yellow in preserved specimens. Mean body length3.06±0.29 mm; range 2.66–3.71 mm (n=10); width 0.57±0.04; range: 0.51–0.65 mm (n=10). Hemicephalic; body orthosomatic, asymmetrically fusiform, widest at mesothorax, somewhat dorso-ventrally compressed (Figure 10). In addition to the three thoracic segments and the nine abdominal segments, there is a pseudosegment, a neck or cervical segment between the head and prothorax. All body segments except neck segment and last two abdominal segments bulge laterally. Penultimate abdominal segment longest and ninth (terminal) segment shortest.Figure 10
The spatulate third instar larva. Note that the head capsule is comparatively minute.Spiracles of prothorax and eighth (penultimate)- abdominal segment prominent, turreted (Figure11). Those on penultimate segment situated laterally in a plica (fold) anterior to the posterior intersegmental suture. Spiracles of remaining segments situated dorsolaterally, and though sessile, annular, and inconspicuous, apparently functional because they are connected to trachea. The tracheae are darkened terminally where attached to peritreme, particularly on sixth and seventh abdominal segments.Figure 11
A turreted spiracle on the penultimate abdominal segment.Body without macropapillae or projections. Also lacking the microdentition seen in first instars. Rather the epidermal surface is minutely verrucose (cuticular verrucae of Mamaev and Krivosheina [14]) on sides and dorsum of all segments, whereas on the ventral surface the microverrucae are confluent and arrayed in transverse lines, presumably as traction ridges. The head is small, retractile, prognathous, the capsule lightly-sclerotized, somewhat conical, anchored with prominent exocephalic tentorial arms. A pair of two segmented (pseudosegmentedcf Solinas et al. [28]) antennae are present, situated subapically, one on each side of mouthparts. Pedicel short, not longer than wide; scape acuminate, fusiform, elongate, length about 3x width. In some images the large placoid sensilla is visible on each scape (Figure 12). The mouthparts are rudimentary and consist of a two-sided labrum, the minute peg-like maxillae, and tiny mandibles. Small, linear, and sclerotized, the latter are aligned parallel to one another and to the sagittal (vertical) axis of the cranium (Figure 14).Figure 12
Head of the third instar. Arrow points to the placoid sensilla on the scape of the antenna.The haired papillae are reduced in number from the first instars (Figure13). The terminal segment has eight stout bristles in two rows of four ranging in length from 10 to 22 μm, the ental pairs longer (mean 14.1±3.1 μm, n=12). Setiform dorsal papillae in a row of six are present on pro- and mesothorax with two on eighth abdominal segments between the spiracles. Their length varies from 22 to 42 μm (mean 32.9±5.5 μm, n=25). All thoracic and first eight abdominal segments have a pair of hind ventrals. Prothorax, mesothorax, and eighth abdominal segments with a pair of pleural setae on each side, remaining segments with one on each side. Outer pleural seta of prothorax longest at 38–50 μm (mean 41.1±5.5 μm, n=10), those on mesothorax next longest at 28–35 μm (32.5±6.4, n=11) with those on the metathorax and abdominal segments shortest at 12–17 μm (24.7±8.3, n=9). Hind ventrals are much smaller (mean 17.1±5.3 μm, n=7). Inner pleural papillae are haired on pro- and mesothorax; longer on prothorax (mean 32.5±3.8 μm, n=4) than on mesothorax (mean 22.3±4.2 μm, n=7) with those on eighth-abdominal segment even shorter at 18 μm. Metathorax and first seven abdominal segments are without dorsal or sternal haired papillae.Figure 13
Diagrammatic position of setiform papillae on the third instar, dorsal on left, ventral on right.Figure 14
Ventral view of third instar head capsule showing rudimentary, paired mouthparts, and the three pronged spatula.The number and position of setiform papillae in Möhn’s [10] account ofLasioptera arundinis are somewhat problematic. He states that there are three pleural papillae on each side of the prothorax but that the third is often unhaired. We find only two onL. donacis. Möhn’s figure of the abdominal segments depict a single pleural seta; yet his text description states there are two, and that is the number that we find inL. donacis as well (Figure 15).Figure 15
Setiform papillae flanking the spiracle on abdominal segment eight: one dorsal and two pleurals on each side.The ventral side of the prothorax has the prominent sclerotized armature referred to as the sternal spatula [29] or breast plate [30], length 0.37±0.04 mm (n=8). Seated on and protruding from the midline it is about ten times longer than greatest width and widest anteriorly where it is anchored to the body by sclerotized lateral apophyses just posterior to the terminus (Figures 14 and 16). The anterior projection is strongly sclerotized, protruding from the body and bearing three forwardly directed teeth: two broad lateral teeth with a smaller middle tooth between and somewhat entad of the lateral teeth. A cross section through the spatula reveals that it is actually hollow. The caudal end of the spatula is expanded into a rounded base, the manubrium, which is embedded deeply in the cuticle. Internally there is a large V-shaped mass of muscle attached to the manubrium which inserts on the dorsum of the prothorax.Figure 16
The three-pronged spatula is flanked by fleshy pads where the apodemes insert. The sternal papillae are seated between the spatula and the neck segment, one on each side (arrows).Attendant to each side of the anterior expansion of the spatula there is a fleshy pad bearing 3 + 2 lateral papillae. The three on the anterior margin are sclerotized and tubular or socket-shaped (Figure18). The outermost of these bears a stout bristle, although in most specimens it is missing, presumably broken off from wear and tear. The two posterior, discal papillae are simple haired papillae (Figure 19). Located on the anterior portion of the prothorax ventrally, between the spatula and the head, there are two conspicuous crateriform structures, one on each side, referred to as the sternal papillae in the taxonomic literature although they are not papilliform (Figures 16 and 17).Figure 17
In profile the sternal papillae appear to have a pair of ducts (arrows) connecting to the paired sensory structures within its crater.Figure 18
The fleshy pads attendant to the spatula have three sclerotized, socket-shaped papillae arrayed on their frontal margin (arrows).Figure 19
On the disc of each fleshy pad is a pair of haired papillae (arrows).Pupa: exarate, enclosed within a silken cocoon; capable of strong movement when disturbed. Body elongate, cylindrical; color off-white in early stages turning to orange later; wing pads black and with darkened patches of scales on the abdominal dorsum in fully mature, pharate specimens (Figures20(a) and 20(b)). Abdominal segments without spines. Length: ranging from 2.2–3.0 mm (mean = 2.55±0.24 mm, n=9). Respiratory horns of thorax slender, tubular basally, filamentous apically. Leg sheaths nonoverlapping, unequal in length; metatarsi attaining abdominal terminus, mesotarsi nearly attaining abdominal segment VIII, protarsi attaining abdominal segment VI. Wing pads overlap metafemora. Beaded antennal sheathes overlap mesofemora and extend just past the base of the wing pads. Palp sheaths extend obliquely anterolaterad, reclining in the space between the eye and profemora. Frons without papillae, horns or ridges. Eyes entire, ocelli absent. Cephalic sclerite bearing a pair of prominent setae each inserted just behind the antennal base.Figure 20
Late stage exarate pupa ofL. donacis, ventral and side views.
(a)
(b)


## 4. Discussion and Conclusions

The larvae of free-living cecidomyiids are variable with respect to the number and size of excrescences on the body surface as illustrated by Peterson [30], Mamaev and Krivosheina [14], and Gagné [11–13]. Such excrescences are typically absent in the endophytic species, includingLasioptera.On the other hand, the form of the body, elongate, cylindrical, and bulging at the thorax (Figure 10), is typical of stem-boring or tunnel inhabiting insect larvae from which one can infer that its morphology facilitates movement within a confined space.The sternal spatula is a characteristic feature of third instar cecidomyiid larvae. Milne [31] found that soil inhabiting species use the spatula to excavate the pupal chamber in the soil. Mamaev and Krivosheina [14] state that phytophagous species use the spatula to perforate or enlarge perforations in plant tissue. Among cecidomyiid species variation occurs primarily in the shape of the apex which can have one, two, or three prongs [14].Lasioptera donacis has a three-pronged spatula. Among the 20 European species ofLasioptera larvae described by Möhn [10], onlyL. arundinis has a three-pronged spatula, the others being cloven with two prongs. A species from Australia,L. uncinata Gagné also has a three-pronged spatula, but with the middle prong the longest and it has only four lateral papillae (five inL. donacis andL. arundinis). Also the host forL. uncinata is a non-graminaceous plant,Melaleuca spp. (Myrtaceae) [17]. According to Gagné [1] none of the New WorldLasiopteraspp. have grasses as the host.Lasioptera arundinis is the species most similar in morphology and ecology toL. donacis. Because the former exploits an endophytic fungus in a closely related grass species, common reed,Phragmites australis (Cav) Trin.,L. donacis andL. arundinis may be closely related species. Or it may be convergence that they share important morphological features in the third instar including a three-pronged spatula and five lateral papillae. The difference between the first instars ofL. donacis andL. arundinis is the presence of long bristles and spines in the latter which relates to a difference in their ecology. According to Rohfritsch [27]L. arundinis lays its eggs outside of the plant under the leaf sheathes of the side shoots. The bristles and spines enable the first instar to carry the fungal conidia which are then disseminated into the plant as the larva feeds on and penetrates the cortical parenchyma of the stem. BecauseL. donacis females lay their eggs along with the conidia into preexisting holes in the leaf sheath surrounding the main stem, the larvae of the latter species do not require such bristles. Also, according to Rohfritsch [4] the first instar larva ofL. arundinis uses its mandibles to perforate the lamella and enter the pith of the stem. The first instar ofL. donacis has minute, rudimentary mandibles.In spite of the reduction in the size of the mouthparts, cecidomyiid larvae, in particular those of the economically important phytophagous species, are noted for the damage inflicted on the host plant. In the case of the Hessian fly,Mayetiola destructor (Say), and the sorghum midge,Contarinia sorghicola(Coquillet), the damage is evidently caused by the injection of digestive fluids rather than by the physical damage by the tiny mandibles [16, 32]. Extraoral digestion is the norm for cecidomyiids whether phytophagous, mycetophagous, or even predaceous [14]. For that purpose the salivary glands are typically enlarged [32]. Our sections ofL. donacis larvae show that the salivary gland dominates the internal thoracic space of the third instar (Figure 22). In cecidomyiids the glands have a dual function: producing digestive fluids during the feeding stage but also producing the silk for the cocoon (Figure 21) in the prepupal stage [33]. We presume that to be the case forL. donacis as well. Apart from having their heads directed at and into the fungal mycelium, we have not observed feeding directly in theL. donacis larvae, presumably because of their light aversion response. Our presumption of extraoral digestion is based on the rudimentary size of the mouthparts and the enlargement of the salivary glands, but also we have observedvomitus when the larvae are placed on filter paper. The lack of accumulations of frass in the colonies, of which residue is typically a conspicuous aspect of insect tunnels, may be a consequence of the mainly liquid diet. Rohfritsch [2] describes feeding byL. arundinis on the endophytic fungus and reports that in combination with extraoral digestion the larva uses the spatula to scrape and scratch the mycelium and then sucks in the solubilized predigested material. The juxtaposition of the spatula to the mouth opening inL. donacis suggests that the spatula serves as an extraoral masticating organ as well (Figure 23).Figure 21
A prepupal third instar makes a cocoon with silk from the labial gland.Figure 22
Longitudinal section of the late third instar shows the tubular salivary gland (arrow) associated with extraoral digestion and silk production.Figure 23
Lateral view of the prothoracic spatula. The head segment with antenna is to the far left. The head is seated in the collar segment within which the dark red Bolwig’s organ can be seen (far right). On the prothorax between the spatula and the collar segment are the sternal papillae, one of which can be seen as a round, tan colored structure (arrow). When the collar segment inverts it brings the mouth into close juxtaposition to the spatula and the sternal papillae.The number and distribution of the minute haired papillae of the first instar ofL. donacis correspond to the chaetotaxy characteristic of the tribe Lasiopterini. In the later instars the number is reduced, especially on the abdomen, while those that persist have been transformed into sensory bristles. Whereas the socketed terminal bristles of the third instars are short and stiff (Figure 24), those on the thorax are long and flexile (Figure 25). In accordance with the nomenclature of Zacharuk and Shields [25] their structure suggests that the terminals are sensilla chaetica, which are tactile in function, while the thoracic bristles are sensilla trichodea, which are chemosensory as well as tactile. The dorsal bristles on the prothorax are arranged in a corona surrounding the head when it is retracted into the neck segment (Figure 26).Figure 24
The terminal papillae of the posterior abdominal segments are stout socketed bristles and appear to be sensilla chaetica.Figure 25
The pleural papillae on the prothorax have the form of flexile socketed setae, that is, sensilla trichodea.Figure 26
Retracting the head into the neck segment juxtaposes the dorsal setae of the prothorax, three on each side ((a), (b), (c)) onto a fold collaring the head.It strikes us that the role of the sensory organs in the endophytic species has been underappreciated. Bolwig’s organ detects light [34] and is presumed to initiate escape and evasion behavior in the event the larvae is exposed. Skuhrava and Skuhravy [35] report that birds break into the cane to predate on the larvae. According to Solinas et al. [28] the gall-inhabiting cecidomyiid larvae have shorter antennae compared to the free living species and that is certainly the case withL. donacis. Whereas the antennae are small and the body is largely devoid of protuberances, the prothorax has conspicuous sensory papillae associated with the spatula. On either side of the frontal attachment of the spatula there is a fleshy pad which has the five lateral papillae. In most described species ofLasioptera there are four except inL. arundinis which also has five in a similar configuration toL. donacis as illustrated by Möhn [10]. Three are situated just behind the anterior margin and are sclerotized and tubular or socket-shaped. Behind them on the disc of the pad are two haired papillae. In the review of the sensory organs of larval insects by Zacharuk and Shields [25] there are no organs which are morphologically analogous to these structures. Being seated on the pads at the insertion of the spatula would suggest that their function may be proprioception related to the operation of the spatula in mastication. Among the postcephalic sensory organs proprioceptors such as the campaniform sensillae are common [36], but they are much smaller than these novel structures.Even more conspicuous are the much larger sternal papillae, one on each side, seated on the ventral side of the prothorax between the spatula and the collar segment. In ectal view they appear to be conical with a pair of internal structures (Figure27). In profile these structures appear to be connected to ducts (Figure 17). Moreover, the structures are eversible; that is, observed in living specimens they sometimes protrude from the central crater as pegs (Figure 28). This peg-in-pit arrangement in accordance with the system of Zacharuk and Shields [25] would be sensilla ampullacea which are chemosensory. The number of pegs varies among larval taxa, as many as seven in some Staphylinidae (Coleoptera) [37]. If they are two-pegged sensilla ampullacea the sternal papillae on the prothorax ofL. donacis are perhaps gustatory in function, that is, an adaptation to extraoral digestion. The juxtaposition of these sensillae near the spatula would perhaps allow the larvae to “taste” the macerated mycelial mass as it feeds. They may thus be functionally analogous to the papilla sensilla found on the terminal organs of the head segment of house fly larva [38]. In the latter study the two pegs are referenced as “dendrites” exposed to the exterior and functioning as taste organs.Figure 27
The sternal papillae in ectal view appear to be conical with paired structures internally (arrows), suggesting that they have a sensory function.Figure 28
The paired structures within the sternal papillae are extrusible and may be sensory pegs (arrows).The reliance on an endophytic fungus for its food resource complicates but does not eliminateLasioptera donacis as a candidate for biocontrol of the invasive weed,Arundo donax. However one can expect that a deeper understanding of the tritrophic relationships among the plant, the fungus, and the insect will be required before a successful program can be implemented.



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*Source: 262678-2015-11-02.xml*