Hydrellia pakistanae Deonier (Diptera: Ephydridae), is a host specific biocontrol agent introduced in the USA in 1987 for the management of Hydrilla verticillata (L.f.) (Royle) (Center et al. 1997). Hydrellia pakistanae is native to Indonesia and has been collected from Pakistan, northern China, and southern India (Deonier 1978). The aquatic larvae damage the plant by mining the leaves during its three larval stadia (Deonier 1971; Freedman et al. 2001). Deonier (1978) indicated that many species in this genus are important herbivores of various species of aquatic plants especially in eutrophic ecosystems. He also indicated that large populations of Hydrellia larvae can impact the biomass of their target host plant, thus influencing littoral plant communities (Deonier 1971). Such is the case for H. pakistanae, where numerous studies have shown that sustained feeding can reduce hydrilla photosynthesis thereby impacting biomass, fragment viability, and tuber production causing shifts in plant assemblages toward increased native plant abundance and diversity (Grodowitz 2000; Freedman et al. 2001; Owens et al. 2008).

Hydrilla is an extremely aggressive submersed freshwater plant that forms thick mats of biomass at the water surface in a variety of freshwater habitats (Langeland 1990). It causes severe ecological and economical damage to aquatic ecosystems by impeding water flow, displacing native plant species, and disrupting boat traffic (Godfrey et al. 1996; Haller 1978). Hydrilla is currently found on every continent except Antarctica, and has become a major problem in waterways around the world (Pieterse 1981). The true origin of hydrilla is still unknown. However, Cook and Lüönd (1982) believed the origin could be traced to the subtropical regions of Asia, where hydrilla is still considered a problem (Pieterse 1981).

The larvae of H. pakistanae are entirely aquatic and, as such, must have evolved specialized structures and/or physiological mechanisms to allow maintenance of internal osmotic conditions when exposed to a highly dilute external medium. However, osmoregulation in this species has not been examined in great detail. To aid in maintaining internal osmotic equilibrium, other species of aquatic insects have evolved specialized internal and external structures (Bradley 1985; Phillips 1981) including chloride epithelia, chloride cells, anal papillae and anal organs (Komnick 1977).

Deonier (1971) noted the presence of 2 plates surrounding the anus in the larva of Hydrellia spp. and identified these structures as anal plates. However, Deonier (1970) neither described the morphology of the anal plates in detail, nor attempted to delineate a function. Information on the anal plates or anal organs in other aquatic dipteran larvae is minimal, but several authors have indicated an osmoregulatory or ion transport function based on specialized staining techniques, electron microscopy, and physiological studies (Stoffolano 1970; Schwantes 1989; Schwantes and Seibold 1989; Reeves 2008). This paper describes the anal plates of Hydrellia pakistanae in detail using both light and scanning electron microscopy; and based on these observations suggests a possible osmoregulatory function.

MATERIALS AND METHODS

Staining Technique

In an attempt to ascribe a function to the anal plates, a modified staining technique was used as described by Stoffolano (1970) and Schwantes (1989). The technique uses a 5% silver nitrate solution in which ionized silver binds with chloride ions to form silver chloride; the latter subsequently darkens upon exposure to light (Komnich 1977; Bradley T. J. 1985). Hence, if a structure darkens after staining it presumably contains higher concentrations of chloride ions and thus indicates a possible osmoregulatory function. The validity of this staining technique for providing evidence for an osmoregulatory function was established by Schwantes & Seibold (1989). Such a staining technique for the detection of ion transport has also been applied to other invertebrates with excellent results (Barra et al. 1983; Kikuchi & Shiraishi 1997).

Larvae of each instar were rinsed in deionized water and then placed in a 5% solution of silver nitrate for 5–10 s. The larvae were then rinsed for 60 s in deionized water and subsequently placed on a glass slide in a small quantity of 5% magnesium sulfate solution (25 ^°C). The magnesium sulfate was used to slightly relax the larvae by interrupting muscle activity thereby assuring exposure of the anal plates. The slide was placed on the microscope stage and exposed to a bright light source. After 5 to 10 min, the ventrally located anal plates were examined for evidence of darkening.

Fig. 1.

Ventral and lateral views of the anal organ of Hydrellia pakistanae. ap = anal plate, an = anus, (a) Ventral view of the unstained anal plates of a first instar with dashed circle around anal plate location. Note the plates and anus are essentially transparent and difficult to discern, (b) First instar with silver nitrate stained anal organs, (c) Second instar with very distinct crescent-moon shaped anal plates, (d) Third instar larva with slightly transparent anal plates. (e) Lateral view of the anal plates of a third instar, not stained, (f) Lateral view of the anal plates of a third instar with a light silver nitrate stain.

RESULTS

Anal Plate Description

The 2 anal plates of H. pakistanae larva are normally transparent to semi-transparent and located on the ventral surface encircling the anus on the 10th larval segment. The anal plates are roughly crescent shaped and appear symmetrical on either side of the anus (Fig. 1b–d). The distinct crescent shapes can be seen in the third instar with the first and second instars having less definition but still maintaining a roughly crescent shape (Fig. 1d). Under light microscopy the plates appear smooth, and lie flat or slightly raised above the body (Fig. 1e, 1f). The anal plates are delineated by a distinct line that is difficult to discern when not stained (Fig. 1A).

Under SEM at lower magnifications the anal plates appear relatively smooth similar to that observed under light microscopy (Fig. 2a). However, at higher magnifications an undulated surface to the anal plates is exhibited (Fig. 2b). Whether this undulating surface appearance is an artifact of the high vacuum conditions associated with the use of living specimens under low vacuum is not known. If high vacuum is the cause, it may indicate a difference in thickness of the cuticle of the anal plates and that of other areas of the body, since the overall body cuticle did not exhibit the same surface change. As under light microscopy, SEM micrographs reveal a distinct delineation in the form of a raised narrow line between the body cuticle and the anal plates (Fig. 2b). At 12,000 X, the cuticle of the anal plate surface appears to have small holes or pits though more detailed electron microscopy is needed to determine the exact nature of these structures separating them from possible artifacts (Fig. 3).

Fig. 2.

SEM micrographs of the anal plates of larval Hydrellia pakistanae. sp = Spiracles, ap = anal plate, an = anus, (a) Overall morphology of the anal plates (b) Close-up of the anal plates from a lateral view. Note the undulating surface appearance under higher magnification which differs from that of the surrounding cuticle.

There are significant differences in the perimeter and surface area of the anal plates with each successive instar (Fig. 4). The anal plates vary in size from about 200 µm to 750 µm in perimeter and 1,800 µm² to 14,000 µm² in area depending on instar. A significant positive correlation (P < 0.05, r > 0.9) exists between anal plate perimeter and surface area with larval body width and length (Fig. 5). These correlations may indicate that the functioning of the anal plates remains constant for all instars. It is likely that if later instars had limited use of the anal plates, the anal plate size would remain small relative to overall body width and length.

DISCUSSION

The most comprehensive review to date of the literature on Hydrellia spp. is that of Deonier (1971). Our morphological description of the anal plates of H. pakistanae expands upon Deonier's initial research, where he noted the presence of anal plates. However, he did not describe the morphology in respect to the size and shape of these organs. We observed the same general findings as Deonier, but with more detail. The distinct crescent moon shaped anal plates apparently are characteristic of H. pakistanae. However, how does the size and shape of the anal plates of H. pakistanae differ from other species of dipteran larva?

Fig. 3.

SEM of the surface of the anal plates of Hydrellia pakistanae at 12,000 X. Note the small pits or possible holes scattered throughout the surface. While these do not appear to be artifacts due to specimen preparation, more microscopy work is needed to characterize them.

Fig. 4:

Total perimeter (µm) and surface area (µm²) for the anal plates for all three instars. Means with different letters are significantly different at P = 0.05.

Stoffolano (1970) described differences among dipteran species in the size and shape of their anal organs. He suggests the differences between the sizes of the anal organ may reflect larval habitat differences, which, through selection, have favored one size organ over the other; larger anal organs are found predominantly in those species that reside in more dilute freshwater habitats. Further, Stoffolano (1970) described the anal plates or anal organs of Musca domestica L. as having lateral arms that are reduced so that the plate is short, squat, and roughly butterflyshaped. In contrast, Musca autumnalis De Geer, a close relative to M. domestica has an anal organ with lateral arms that are large and that wrap around 3/4th of the circumference of the posterior end of the larva. Musca autumnalis resides in fresh manure from cows (Bos spp.; Artiodactyla: Bovidae) fed pasture grasses, a larval habitat that is relatively low in dissolved ions (i.e., osmolality typically < 200 mmol/kg); i.e., freshwater (Grodowitz 1985). In contrast, M. domestica lives in a wide variety of habitats, but is more successful in those that are higher in dissolved ions; i.e., having a higher osmolality, such as chicken (Gallus gallus domesticus (L.); Galliformes: Phasanidae) feces, which are a mixture of feces and urine. Stoffolano (1970) examined figures from Zimmin (1951) of the anal plates of Muscidae and noted that besides differences in anal organ size, there is much variation in surface appearance and shape. For example, the larvae of M. domestica have smooth flat plates, while others like Stomoxys calcitrans (L) have bulbous or “hemorrhoidlike” surface structures. Based on Stoffolano–s work, the relatively small size of the anal plates of H. pakistanae is not characteristic for dipteran larvae, like 77. pakistanae, which thrive in highly dilute freshwater environments. Reasons for this discrepancy are not fully understood, and more research is needed especially using more detailed transmission electron microscopy and other physiological experimentation.

Fig. 5:

Graph of larval surface area (log₁₀) versus larval length (log₁₀) for all instars combined. Correlation was significant at P < 0.05 with r values of 0.95 with a sample size of n = 30. Note the distinct groupings for each instar indicating increasing size of the anal organ for each successive instar.

The images H. pakistanae larvae using SEM photography documented 2 possible artifacts due to exposure to high vacuum conditions and rapid desiccation. First was an unusual undulating pattern to the entire body of the larvae which was even more pronounced for the anal plate cuticle. The first artifact is most likely due to the vacuum environment of the SEM chamber and subsequent desiccation of the specimen. However, the more pronounced ‘wrinkling’ of the anal plate cuticle may indicate that it is thinner since it has a higher degree of shrinking than the rest of the body cuticle. Stoffolano's (1970) work on dipteran larvae histological sections of the anal plate cuticle showed the plates to be thinner than the rest of the body cuticle. This supports the idea that the higher degree of shrinking seen in H. pakistanae anal plate cuticle is a result of a thinner cuticle.

Stoffolano also noted spherical structures underneath the anal plate cuticle of several species of dipteran larvae much like our observations of small pits or holes in H. pakistanae. He described these structures as large epidermal cells which contained polytene chromosomes that gave a cobble-stoned appearance to the cuticle. More morphological studies of these spherical structures, especially using transmission electron microscopy, may be necessary to further describe their morphology, and to ascribe a function of the small pits.

Many scientists have used the silver nitrate staining method as a reliable tool to ascribe the function of osmoregulation to the anal plates or anal organs of many dipterans (Simmin 1951; Stoffolano 1970; Komnich 1977; Bradley 1985; Schwantes 1989; Schwantes & Seibold 1989; Reeves 2008). The anal plates of H. pakistanae stain a dark brown after immersion in a weak solution of silver nitrate and subsequent exposure to high light intensity. This indicates the presence of high concentrations of chloride ions within the anal plate tissues. The presence of high chloride concentrations implies an osmoregulatory function; and similar staining techniques have been used to ascribe osmoregulatory functions to tissues in a wide range of aquatic or terrestrial insects (Komnich 1977; Bradley 1985). Such chloride concentrating epithelium is typically found in freshwater organisms that must have a mechanism to maintain internal osmotic pressures when exposed to an external medium which is highly dilute relative to internal osmotic pressures. Based on Stoffolano's (1970) work, the presence of osmoregulating anal plates in H. pakistanae is typical of dipterans residing in a low salinity environment (fresh water) that, therefore, have a need to maintain internal osmotic conditions at a higher level than their surrounding habitat.