THE WASMANN JOURNAL OF BIOLOGY 47(1-2), 1989, pp. 1ll-126 ULTRASTR UCTURE OF THE MALPIGHIAN TUBULES WITH ENLARGED SEGMENTS IN THE ALKALI E PHYDRA HIANS SAY FROM , CALIFORNIA

Helen Yu, Eduardo A. C. Almeida a nd PaulK. Chien

Abstract.-The fourth inst ar larvae of the Mono Lake alkali fl y hians has two pairs of dissimilar Malpighian tubules (MT). The one studied here has enlarged segments that contain spherical mineral concretions. The MTs extends from between the mid and hind gut through a common ureter, which branches into two proximal segments. The ureter and proximal seg­ ment (about 90 ,urn in diameter) cells are ultrastructurally similar. Both conta in an elaborate basal labyrinth, intracellular electron dense vesicles, numerous m itochondria, glycogen granules, rough endoplasmic reticulum (RER) and a brush border wi th microvilli penetrated by mitochondria. The enlarged segm ent, about 350 .urn in diameter, is continuous with the proximal segment. The celJs of the enlarged segment are flattened, contain mitochon­ dria, RER, an ela borate basal labyrinth, and numerous m icrovilli. The lumen of the enlarged segment contains large quantities of concretions. This seg­ ment graduall y decreases in diameter to become the transition segment, wh ich bends and becomes the long main segment (about 90 ,urn in diameter). The terminal blind end of the main segment attaches to the rectum. The ultrastructure of the transition segment is intermediate between the main and enlarged segments. The celJs in the main segment exhibit welJ developed basal labyrinth, numerous mitochondria, and electron dense mineral de­ posits within vesicles between 0.3 and 0.5 .urn in diameter. In addition, RER and microvilli containing mitochondria are present. No accessory cells were fo und in MT from the fourth instar larvae.

Introduction The larva of the alkali fly Ephydra hians is the major benthic metazoan found in Mono Lake, California. Mono Lake water is characterized by high osmolarity (1,900 mOsm) and extreme alkalinity (pH 10.5) due to its high carbonate a nd bicar bonate content. The osmotic concentration of the he­ molymph of the larvae (determined by the vapor pressure method) is 430 mOsm, and its pH is about 7.2 (Yu eta/., 1988). The fl y larvae that inhabit the lake must possess mechanisms to regulate both their hemolymph con­ centration and pH. Malpighian tubules in are believed to be the major organ in hemolymph osmoregulation. In E . hians, one of the two pairs of Malpighian tubules produce and store numerous spherical mineral concretions that are mostly composed of calcium carbonate (Yu eta/., 1988). 112 THE WASMANN JOURNAL OF BIOLOGY

The general morphology and ultrastructure ofMalpighian tubules in other dipterans that produce mineral concretions, such as Drosophila melanogas­ ter, Drosophila hydei and Ephydra riparia, have been described by Wessing and Eichelberg (1975), in D. hydei by Revert (1975), and Wolburg et al. (1973), and in the brackish water mosquito Culiseta inornata by Garrett and Bradley ( 1984). The major morphological and ultrastructuraJ features of the Ma1pighian tubules of these dipterans are similar to those observed in E. hians. However, some differences exist. Drosophila spp., and E. riparia have distal en larged segments and proximal main segments (Eichel berg and Wess­ ing, 1975). This arrangement is reversed in E. hians. The proximal segments of the Malpighian tubules in E. hians are enlarged while the distal main segments are long and narrow (Yu et a/., 1988). The Malpighian tubules of the blowfly Calliphora erythrocephala (Berridge and Oschman, 1969) show many similarities to those of E. riparia. Accessory cells in the Malpighian tubules main segments have been found in both species (Eicbelberg and Wessing, 1975). The species that shows the most similarities at the gross morphological and ultrastructural level to E . hians is E. riparia, as described by Eichel berg ( 1979). However, because E. riparia is a eurybaune dipteran inhabiting coastal salt water marshes (Sutcliffe, 1960) as opposed to the alkaline environment which this E. hians inhabits, the specific adaptation of the Malpigbian tubules of E. hians may be quite different from E. riparia. This work describes the morphology and ultrastructure of the Malpighian tubules with enlarged segments in the alkau fly E. hians collected in Mono Lake, California.

Materials and Methods Larvae of the alkali fly Ephydra hians were coUected from Mono Lake shores and kept at room temperature (Yu et a/., 1988). The fourth instar larvae, I 0 mm or greater in length were used in this study. For transmission electron microscopy (TEM), Malpighian tubules were dissected and fixed at room temperature for I hr in 5% glutaraldehyde (GA) in 360 mM cacodylate buffer, pH 7.5. The tissue was postfixed in I% osmium tetroxide. Samples were dehydrated in an acetone series and embedded in low viscosity epoxy resin (Spurr, 1969). Sections were obtained with a diamond knife, stained with uranyl nitrate and lead citrate (Reynolds, 1963) and photographed using a Zeiss EM-9S2 electron microscope. For freeze-fracture, Malpighian tubules were dissected in 25% glycerol in 360 mM cacodylate buffer at pH 7.5 and immediately frozen in liquid freon cooled by liquid nitrogen at - 170°C. The specimens were fractured at - l00°C and etched for 3 minutes in a Balzers freeze fracture apparatus. Platinum replicas were carbon stabilized, cleaned with 4% sodium hypochloride followed by I N HCI and supported on form­ var coated copper grids. VOLUME 4 7, NUMBERS I AND 2 113

MALPIGHIAN TUBULES WITH ENLARGED SEGMENTS ANTERIOR END : DORSAL SURFACE ..

POSTERIOR END

SEGMENT 2mm VENTRAL SURFACE Figure I. Diagrammatic representation of the pair of Malpighian tubules with enlarged segments.

Results The Malpighian tubules of the fourth instar larvae of E. hians are about 13 mm in length. The Malpighian tubules of a I 0 mm larva have a ureter and proximal segment which makes up about 10% of the total length. The enlarged segment of the tubule comprises about 20% of the total length, while the transition segment represents about 20% and the main segment 50%. The pair of Malpighian tubules with enlarged segments (Fig. 1) branches from the ureter at the junction between the midgut and hindgut. The ureter (about 90 ,urn in diameter) splits into two short proximal segments which increase in diameter and become the enlarged segments. The enlarged seg­ ments, 350 ,urn in diameter, extend ventrally and toward the anterior. The enlarged segments decrease in diameter gradually to form the transition segment (180 ,urn in diameter). The transition segment bends posteriorly at the region marked by the presence of surrounding yellow fat bodies. The main segment measures about 90 ,urn in diameter and continues from the transition segment toward the posterior end of the gut, where its blind end attaches to the wall of the rectum. Small concretion cores (about 0.3 ,urn) containing mostly calcium and phosphorus with some magnesium (Yu et a/., 1988) are produced in and secreted from the cells throughout the length of the main segment. Concretions with a diameter up to 21 ,urn were found in the lumina ofthe main segment, transition segment, and enlarged segment. The concretions can be eventually discharged into the gut, a process con­ trolled by the musculature around the proximal segments and ureter. Freeze-fracture of cells from the main segment (Fig. 2) shows a brush border with two types of microvilli. One is penetrated by long, slender 114 THE W ASMANN JOURNAL OF BIOLOGY

Figure 2. Freeze-fracture electron micrograph of a Malpighian tubule main segment in cross section. C, concretions; BL, basal lamina; M, mi tochondria; MI, mitochondrion within mi­ crovillus; MV, microvilli. VOLUME 47 , NUMBERS I AND 2 115 mitochondria measuring 0.2 JLm to 0.3 JLm in diameter, while the other type of microvilli are about 0.15 JLm and do not contain mitochondria. In the cytoplasm, numerous spherical vesicles containing mineral concretions can be observed. The size of the vesicles varies from 0.1 JLID to about 4 JLm, most of them range between 0.3 and 0.5 JLm. Many mitochondria are also present in the cytoplasm. Mineral concretions are also found between the basal folds in the basal lumen. Transmission electron micrographs from cells of the main segment show intracellular electron dense mineral deposits (Figs. 3 and 4). The brush border is similar to that observed in the freeze fracture preparation, with the two types of microvilli. However, the basal folds appear enlarged and lack the mineral concretions seen in freeze-fracture replicas. The basal lamina can be clearly distinguished in these transmission electron micrographs. Higher magnification of the cytoplasm reveals well-developed endoplasmic retic­ ulum in addition to numerous mitochondria and concretions (Fig. 4). The basic ultrastructure of the transition segment cells (Fig. 5) is very similar to that of the main segment (Figs. 2-4) cells. However, there are considerably less vesicle bound electron dense deposits. The brush border in the transition segment also shows two types of microvilli, one with mi­ tochondria another without (Fig. 6). Concretions can be seen nested in the brush border (Fig. 6). In this segment, the diameter of the Malpighian tubule increases relative to the main segment while the cells become thinner (Fig. 7). The Malpighian tubule attains maximum diameter in the enlarged segment (Fig. 1), while the cell thickness is at a minimum (Figs. 8 and 9). The cytoplasm (Fig. 8) has numerous mitochondria. Other organelles such as rough endoplasmic reticulum and golgi apparatus are also present but are less prominent than in the cells of the main segment. The majority of the microvilli in the brush border do not contain mitochondria. Luminal con­ cretions of varying sizes can also be seen nested among the microvilli (Fig. 9). There are very few cytoplasmic concretions in this region. However, there are membrane bound vesicles containing possible remnants of extracted mineral concretions in an extensive system of basal folds (Figs. 8 and 9). Freeze-fracture of the enlarged segment shows well preserved mineral con­ cretions within the basal lumen (Fig. 10). The proximal segments of the Malpighian tubules are continuous with a common ureter (Fig. 1) that discharges concretions into the midgut. The cell ultrastructure of the proximal segment of Malpighian tubule is similar to that of the ureter. The ureter, however, is surrounded by more muscle fibers. The cells of the proximal segment (Fig. 11) show numerous mitochondria, well-developed rough endoplasmic reticulum, glycogen granules and cyto­ plasmic concretions that are partly extracted. The majority of microvilli in the brush border (Fig. 12) contain mitochondria. The basal folds are different in appearance from those in all other regions showing a fine reticulation (Fig. 116 THE WASMANN JOURNAL OF BIOLOGY

; MV~

• ·~

3

BL 1.0pm .1 - 4 VOLUME 47, NUMBERS I AND 2 117

13). Muscle fibers (Fig. 13 and 14) are found adjacent to the basal lamina of these cells.

Discussion The Malpighian tubules ofthe alkali fly E . hians have many morphological and ultrastructural elements in common with those of other dipterans, name­ ly Drosophila spp. and E. riparia. Basic anatomical structures such as the ureter, the main segment, and enlarged segment and the transition segment have been described in other dipterans such as those discussed above but they are arranged differently. In Drosophila spp. and E. riparia (Eichelberg and Wessing, 197 5) like in E . hians there are two pairs ofMalpighian tubules, one of which has enlarged segments. However, E. hians has proximal en­ larged segments and distal main segments (Yu et af., 1988) while Drosophila spp. and E. riparia have a reversed arrangement with distal enlarged seg­ ments and proximal main segments (Eichel berg and Wessing, 197 5). Fur­ thermore, because of the uniqueness of the carbonate/bicarbonate rich al­ kaline environment of E. hians, the Malpighian tubules must function differently from those of other dipterans. The larvae offruit , Drosophila spp., live in decomposing organic matter and produce mineral concretions different in inorganic composition (Hevert eta!., 1974) to that of the alkali fly, (Yu eta!., 1988). The habitat of E . riparia, coastal salt marshes, (Sutcliffe, 1960), is rich in sodium chloride, leading to the formation of concretions with different mineral composition. In particular the concretions found in D. hydei seem to be mostly composed of calci urn and phosphate (Hevert et al., 1974), a composition comparable to the cores ofconcretions in E . hians, however they contain no more than 5% carbonate/bicarbonate, unlike the cortex of concretions in E . hians which has mostly calcium carbonate. The Malpighian tubules with enlarged segments in the alkali fly larva E. hians have five morphologically distinct regions. Distal relative to the gut, and attached to the wall ofthe rectum is the blind end of the main segment. The main segment is where the flow of solid concretion "urine" starts. This segment comprises about 50% of the total length of the Malpighian tubule, and throughout its cells we find numerous vesicles with electron dense de­ posits, presumably the cores of mineral concretions before secretion into the

Figure 3. Electron micrograph of a Malpighian tubule main segment in cross section. C, concretions; BL, basal lamina; M, mitochondria; MI, mitochondrion within microvillus; MV, microvilli. Figure 4. Electron micrograph of the cytoplasm of the Malpighian tubule main segment. RER, rough endoplasmic reticulum; C, concretions; BL basal lamina; M, mitochondria. 118 THE WASMANN JOURNAL OF BIOLOGY

lumen. These cores have been demonstrated by us (Yu eta/., 1988) to have a different composition than that of the concretion cortex that will form in the Malpighian tubule lumen. The core contains mostly calcium and phos­ phate with some magnesium while the cortex will have calcium and car­ bonate/bicarbonate. The transition segment follows and it is appropriately named because it represents both a gross anatomical transition between the narrow main segment, and the wide enlarged segment, and also a transition of ultrastructure between the two types of cells. The enlarged segment has very Aat cells with few or no intracellular mineral deposits. This region is not thought to function in the secretion of concretion cores, but rather in other unknown functions. Inside the enlarged segment very large numbers of concretions are stored. The passage of the concretions into the gut is controlled by the narrow short proximal segments and common ureter that connect the enlarged segment to the gut. The proximal segments are sur­ rounded by muscle fibers that can constrict the lumen and block passage into the gut, and by tracheoles that presumably provide oxygen to muscle celJs in this region. The proximal segment cells also store glycogen, possibly as an energy source for the function of the striated muscle cells that surround the proximal segments and the ureter. The ureter shows an ultrastructural transition between the proximal segment and the gut. The epithelial cells of the main segment and enlarged segment show typical characteristics of transport celJs. However the distribution of mitochondria, the convolutions of the basal labyrinth and the elaboration of the brush border vary from one region to the other. The cells of the MT main segment contain mitochondria, predominantly in numerous elongated microvilli while in the flattened sac cells, the mitochondria are absent from the microvilli. Maio segment cells have numerous mitochondria, but not as many as can be found in the cytoplasm and near the basal cell membrane of enlarged

Figure 5. Electron micrograph of the Malpigbian tubule transition segment. C, concretions; CR, concretion remains; DL, basal lamina; M., mitochondria; MI, mitochondria within micro­ villi. Figure 6. Freeze-fracture replica of the brush border in the transition segment. MV, mi­ crovillus; MI, mitochondrion within microvillus. Figure 7. Electron micrograph of the cytoplasm of the transition segment. C, concretions; M, mitochondria; Ml, mitochondrion within microvillus; MV, microvillus. Figure 8. Electron micrograph of the cytoplasm of the enlarged segment. CR, concretion remains; DL, basal lamina; M, mitochondria; MV, microvilli. Figure 9. Electron micrograph of the enlarged segment. C, concretion; CR, concretion re­ mains; DL, basal lamina; M, mitochondrion; MV, microvilli. Figure 10. Freeze-fracture replica of the basal folds in the enlarged segment. C, concretion. 120 THE WASMANN JOURNAL OF BIOLOGY

· ~-

CR ' VOLUME 47, NUMBERS I AND 2 121

c '\• 9 122 THE WASMANN JOURNAL OF BIOLOGY

.. ' .

... ..

~ EBL

~ MBL

~ MU

.... 11 VOLUME 47, NUMBERS I AND 2 119 VOLUME 47, NUMBERS I AND 2 123 segment cells. This difference in polarity and distribution of mitochondria may be an indication of the location of ion transport activity. In the enlarged segment cells, the microvilli are poorly developed compared to the other regions of the MT. The cells of the enlarged segment have a basal lumen that appears swollen and extends into the cell. Cells in the main segment are characterized by the presence of numerous electron dense mitochondria both in the cytoplasm and in the microvilli; however, some mitochondria are observed among the basal infoldings. This is not the case in D. melanogaster (Eichelberg and Wessing, 1975) where the distribution of mitochondria in the main segment cells show distinct polarity, with the mitochondria concentrated either at the apical or the basal regions of the cell. The polarity seen in E. hians seems to indicate that cell membrane transport in the main segment is predominantly located in the luminal brush border while in the enlarged segment thjs localization is more promjnent in the basal labyrinth. A second type of cells that has poorly understood functions can often be found in the Malpighian tubules of dipterans. These cells are named acces­ sory or stellate cells. Accessory cells were reported in the main segment of the Malpighian tubules in the larva of E . riparia (Eichelberg and Wessing, 1975). These cells occupy about 5% of the tubule cross sections and differ in ultrastructure from the main cells. The microvilli of the accessory cells are reduced in number and size with no mitochondria. In addition, there are no transport vesicles with electron dense mineral deposits in the cyto­ plasm of these cells. In the fourth larval instar of E. hians, no accessory cells were found. However, we observed them in the third instar larval stage. According to Eidmann and Kuhlhorn (1 970), accessory cells may be regen­ erating cells, which replace the main cells during metamorphosis. Ifwe accept tills hypothesis, the absence of the accessory cells in the fourth, which is also the last, instar in E. hians may be due to eminent pupation. The stellate (accessory) cells in the Malpighian tubules of Calliphora erythrocephala show affinity for lead ions which can be visualized by precipitation with ammo­ nium sulfide (Berridge and Oschman, 1969). As a result, Berridge and Osch­ man (1969) suggested that the accessory cells are not only structurally dif­ ferent but also functionally different from the main cell type. However, in E. hians, affinity for lead was not observed by us in any cells of the fourth or third instar Malpigruan tubules. Given the lack of affinity for lead by the

!- Figure II. Electron micrograph of the proximal segment. MU, muscle fiber; MBL, muscle cell basal lamina; EBL, epithelium basal lamina; RER, rough endoplasmic reticulum; CR, concretion remains; M, mitochondria; MI, mitochondria within microvilli. 124 THE WASMANN JOURNAL OF BIOLOGY

# 12 ,.....

13 VOLUME 47, NUMBERS I AND 2 125

Malpighian tubule cells, it is possible that the accessory cells in third instar E. hians may be of the regenerative type. Ephydra hians is a dipteran with unique adaptations to ion and osmo­ regulation in lake water of high alkalinity and salinity. The gross morphology and fine structure of the Malpighian tubules of E . hians show general sim­ ilarities to those found in other dipterans. However, the Malpighian tubules in E. hians are adapted to function in alkaline and hypersaline conditions and consequently are functionally and structurally different from those of other organisms previously studied.

Literature Cited

Berridge, M. J., and J. L. Oschman. 1969. A structural basis for fluid secretion by Malpighian tubules. Tissue and Cell, 1:247-272. Eichel berg, D. 1979. Ultrastructure and experimental modifiability of Malpighian tubules in larvae of the salt fly Eph.ydra riparia (Diptera: ). Entomologica Germanica, 5(4):301 -315. Eichelberg, D., and A. Wessing. 1975. Morphology of the Malpighian tubules of insects, pp. 124-147. In A. Wessing, Excretion. Gustav Fischer Verlag, Stuggart. Eidmann, H., and F. Kuhlhorn. 1970. Lehrbuch der Entomologie. Paul Parey Verlag, Ham­ burg-Berlin. Garrett, M. A., and J. T. Bradley. 1984. Ultrastructure of osmoregulatory organs in larvae of the brackish water mosquito Culiseta inornata. Journal of Morphology, 182(3):257-278. Hevert, F. 197 5. Physiologische mechanismen bei der harnbildung durch Malpighische gefabe. Fortschritte der ZooIogie, 23: 173-192. Hevert, F., H. Wolburg, and A. Wessing. 1974. Die Konkremente des larvalen Primarharnes von Drosophila hydei. n. Die anorganischen Bestandteile. Cytobiologie, 8:312-319. Reynolds, E. S. 1963. The use oflead citrate at high pH as an electron-opaque stain in electron microscopy. Journal of Cell Biology, 17:208-212. Spurr, A. R. 1969. A low viscosity epoxy resin embedding medium for electron microscopy. Journal of Ultrastructural Research, 26:31-43. Sutcliffe, D. W. 1960. Osmotic regulation in the larvae of some euryhaline diptera. Nature, 187:331-332. Wessing, A., and D. Eichel berg. 1975. Ultrastructural aspects of transport and accumulation of substances in the Malpighian tubule, pp. 148-172.ln A. Wessing, Excretion. Gustav Fisher Verlag, Stuggart. Wolburg, H., F. Revert, A. Wessing, and J. Porstendorfer. 1973. Die konkremente des larvalen primarharnes ve Drosophila hydei. I. Struktur. Cytobiologie, 8:25-38.

Figure 12. Electron micrograph of the proximal segment cell cytoplasm and brush border. RER, rough endoplasmic reticulum; M, mitochondria; Ml, mitochondria within microvilli. Figure 13. Electron micrograph of the proximal segment cell basal cytoplasm. RER, rough endoplasmic reticulum; M, mitochondria; EBL, epithelial cell basal lamina; MU, muscle fiber; MBL, muscle cell basal lamina. Figure 14. Electron micrograph of the muscle fiber in cross section. MU, muscle fiber. 126 THE WASMANN JOURNAL OF BIOLOGY

Yu, H ., E. A. C. Almeida, P. J. Schulz, and P. K. Chien. 1988. Electron microscopic and x-ray elemental analysis of spherical mineral concretions in brine fly larva Ephydra hians Say from the alkaline Mono Lake, California, 46(1-2):49-65.

(HY and PKC) Department of Biology, University of San Francisco, San Francisco, CA 94117-1080; (EACA) Department of Zoology, University of California, Davis, Davis CA 95616.