The Occurrence and Fate of the Meconium and Meconial Peritrophic Membranes in Pupal and Adult Mosquitoes (Diptera: Culicidae)

Abstract

In mosquitoes, in addition to larval and adult peritrophic membranes (PMs), a PM (meconial peritrophic membrane or MPM1) forms in the pupa around the meconium, the sloughed, degenerating larval midgut epithelium. Often, a second membrane (MPM2) forms in temporal proximity to adult emergence. Differences in the occurrence, persistence, and timing of disappearance of the meconium/MPMs and gas were studied by dissecting the midgut contents from pupae of known ages postpupation and from adults of known ages postemergence. MPM1 was found in all Anopheles and Culex studied and nearly all Culiseta. The occurrence of MPM1 varied in the Aedes species. In one series of Aedes aegypti (L.) dissections, no fully formed MPM2 was found in any specimens. The occurrence of MPM2 appeared to be associated with adult emergence and varied among and within the seven species studied. It typically was seen in recently emerged adults but was observed occasionally in old pupae. Much of our data supports the idea that MPM2 formation is stimulated by midgut epithelium distention.

Culicidae, meconium, metamorphosis, mosquito, peritrophic membrane

The peritrophic membrane or matrix (PM) has been described in numerous arthropods as a protein-carbohydrate matrix with embedded chitin fibrils that separates the midgut epithelial cells from ingested food (Jacobs-Lorena and Oo 1996, Lehane 1997). This membrane is of particular interest in vector biology because of its possible role as a barrier to potentially pathogenic microorganisms ingested with food.

Two types of PM are recognized by their method of formation. Type I PMs are formed by secretions from cells distributed over the entire midgut surface and are found in blood-feeding insects such as black flies, mosquitoes, sand flies, and tabanids (Reid and Lehane 1984, Lehane 1991). Type II PMs are produced by the cardial epithelial cells, which form the outer layer of the proventriculus (Wigglesworth 1929, 1930). All larval Diptera, including adult tsetse flies, hippoboscids, and biting muscids, produce a type II PM (Lehane 1991).

There has been little study of stimulation of PM formation. In adult mosquitoes, the secretion of type I PM appears to be triggered by the physical distention of the midgut epithelium associated with a blood meal (Freyvogel and Jaquet 1965). Type II PM in mosquito larvae appears to be secreted continuously, independent of food ingestion (Becker 1978, Jacobs-Lorena and Oo 1996).

In mosquitoes, a PM has been described in the pupal stage in addition to the larval and adult female PMs (Romoser and Rothman1973, Romoser 1974, Moncayo and Romoser 1994). A PM (meconial peritrophic membrane, MPM1; Figs. 1 and 2) forms around the meconium, the sloughed, degenerating larval midgut epithelium (Romoser and Rothman 1973, Romoser 1974, Lerdthusnee 1986, Moncayo and Romoser 1994). A second membrane (MPM2; Fig. 2) sometimes forms around the MPM1/meconium in temporal proximity to adult emergence. Both MPM1 and MPM2 are type I. MPMs may wall off potentially harmful microorganisms present in the gut lumen from the larval stage and also may protect the developing adult midgut epithelial cells from hydrolytic enzymes associated with meconial degeneration (Moncayo and Romoser 1994).

Fig. 1

Meconium within the posterior midgut of an adult female An. punctipennis, 0–4 h postemergence. Width of the meconium ranges from 100 to 150 μm along its longitudinal axis. Midgut epithelium is breaking down as a result of dissection in distilled water. Me, meconium; MPM1, meconial peritrophic membrane; MgEp, midgut epithelium; Ga, gas bubble.

Fig. 2

Meconium dissected from the midgut of a female adult An. punctipennis, 0–4 h postemergence. Me, meconium; MPM1, meconial peritrophic membrane; MPM2, meconial peritrophic membrane 2.

The current study of several mosquito species compares the timing of the appearance of MPM1 during the pupal stage, the fate of MPM1 and the meconium, and the occurrence and fate of MPM2 in newly emerged adults.

Materials and Methods

Species Studied and Rearing Procedures

Anopheles punctipennis (Say), Culex pipiens pipiens L.(for adult dissections), Aedes triseriatus (Say), and Culiseta impatiens (Walker) were collected in Athens County, OH, and reared in water from their natural habitats or in distilled water. Aedes aegypti L., Rockefeller strain, An. stephensi Liston, Cx. pipiens quinquefasciatus Say, and Cx. p. pipiens L. (for pupal dissections) were obtained from the U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, and raised from eggs in distilled water. Larvae were fed a 1:1:1 mixture of liver powder, ground laboratory chow, and brewer’s yeast. Larvae, pupae, and adults were maintained at 27 ± 1°C.

Pupal and Adult Dissections

Pupae were held in individual plastic cups until the age at which they were dissected. For dissection, adults were anesthetized by placing them in small vials in a bucket of crushed ice.

Dissections were done at room temperature using a dissecting microscope. Midguts were removed using finely sharpened watchmaker’s forceps, placed in phosphate-buffered saline (PBS) or Drosophila Ringer’s, and examined using a compound microscope. Pupae of three species were dissected at selected times after pupation: Ae. aegypti, 0–15, 15–24, 24–30, 30–40, and 40+ h; Cx. pipiens pipiens and An. stephensi, 0–4, 4–8, 8–12, 12–16, 16–20, 20–24, 24–28, 28–32, and 32–36 h.

Midgut contents were described on the basis of the following: presence/absence of gas, presence/absence of a meconium, presence/absence of MPM1 and/or MPM2, and extent of formation of MPM1 and/or MPM2.

Results

Pupae

Results of pupal dissections are shown in Table 1. In Ae. aegypti females, MPM1 was first observed in the 15–24 h postpupation group and persisted in the vast majority of the 40+ h group. MPM2 was not observed in any of the specimens before pupal-adult ecdysis.

Table 1

Timing of occurrence of meconial peritrophic membranes (MPM1 and MPM2) in pupae

In An. stephensi, meconial PM1 first appeared in both females and males in the 8- to 12-h group and was seen in all specimens in the 12- to 16-h and older groups. MPM2 was seen in individuals before as well as after pupal-adult ecdysis.

In Cx. p. pipiens, meconial PM1 was seen in all specimens of both sexes in the 8- to 12-h and older groups. Meconial PM2 was first seen in males in the 28- to 32-h group and in females in the 32- to 36-h group.

Adults

Adult dissections (Table 2) revealed differences in the occurrence, persistence, and disappearance of the meconium and MPMs. MPM1 was present in all Anopheles and Culex adults, and all but one Cs. impatiens. However, not all meconia in newly emerged Ae. aegypti and Ae. triseriatus adults were surrounded by MPM1. In these cases, the meconia were found as loose, green particles within the adult midgut that often dispersed upon dissection.

Table 2

Timing (hours after emergence) and frequency of occurrence [%(n)] of meconial peritrophic membranes (MPM 1 and 2), meconium, and gas in adult mosquitoes

MPM2 was seen often as a well-defined membrane around MPM1, and appeared more separated from the meconium. In many instances, gas bubbles were seen within the confines of MPM2. The timing of MPM2 appearance varied in adults (Table 2). It was first seen in the 1- to 2-h group in Ae. aegypti; the 0- to 4-h group in Cx. p. pipiens,Cs. impatiens, and An. punctipennis; the 4- to 6-h group in An. stephensi; and the 4- to 8-h group in Ae. triseriatus and Cx. p. quinquefasciatus. In Ae. aegypti and Ae. triseriatus, MPM2 often was formed only partially. In this case, the membrane seemed to be formed partially in the posterior region of the midgut.

In all species studied, midgut gas disappeared during the first 24 h after emergence. The earliest disappearance occurred in the two Aedes species at 8–12 h after emergence. In An. punctipennis and the two Culex species, gas was not seen after the 12- to 16-h postemergence interval. In Cs. impatiens, gas was last seen 16–20 h after emergence.

The timing of meconium/MPMs disappearance ranged from 16 to 20 h postemergence in An. punctipennis and Ae. aegypti to the 24 h or more postemergence groups in Cx. pipiens pipiens,Ae. triseriatus, and Cs. impatiens.

No differences were seen between males and females in the occurrence, nature, persistence, and fate of the meconium/MPMs.

Discussion

Although no major differences were detected between males and females, differences in the occurrence, persistence, and disappearance of the meconium/MPMs and gas were observed among the species used in this study. Meconium was found in all pupae and newly emerged adults and MPM1 was found in all specimens of Anopheles and Culex and nearly all of the Culiseta (97%). However, the occurrence of MPM1 in the Aedes species was variable, 24.3% in Ae. triseriatus and ranged from 21.9 to 77.1% in Ae. aegypti. The Rockefeller strain of Ae. aegypti is a long-standing, laboratory strain and it is possible that selection has occurred in association with a relatively uniform diet and rearing environment, resulting in decreased MPM production. However, all specimens of Cx. p. quinquefasciatus, also from a long-standing laboratory strain, had a well-formed MPM1. Furthermore, Ae. triseriatus, a member of the same Aedes genus, but field-collected in our study, also showed decreased MPM1 production. Therefore, the lack of consistent MPM1 production could be a characteristic of the Aedes species studied, and not because of the differences in rearing environment. The fact that MPM1 is formed in some individuals and not in others raises the interesting possibility that its formation may be inducible in response to some cue or cues, perhaps of microbial origin, within the developing midgut.

The occurrence of MPM2 also varied among, and within, the seven species studied. Only Ae. aegypti,An. stephensi, and Cx. pipiens were studied as pupae. Among these species, a membrane interpreted as MPM2 was found in a several An. stephensi pupae and a few Cx. pipiens pupae, which were almost ready to emerge as adults. In adults, MPM2 was seen as early as 0- to 4-h postadult emergence in An. punctipennis,Cx. pipiens, and Cs. impatiens. In Cx. quinquefasciatus and Ae. triseriatus dissections, MPM2 first appeared around the meconium in 4- to 8-h specimens. In one series of Ae. aegypti dissections, no fully formed MPM2 were found in any specimens. However, in this group, a partially formed MPM2 was sometimes seen even in the absence of MPM 1. This membrane was first observed at 4- to 8-h in dissections of the posterior region of the midgut. In the other Ae. aegypti dissections, the MPM2 was seen in 4/61 specimens. In cases of apparent partial MPM2 formation, it was possible that the mosquito was dissected while MPM2 was forming and it adhered to the midgut epithelium in the anterior region.

Romoser (1974) described MPMs in Ae. aegypti,Ae. triseriatus,An. quadrimaculatus,Cx. nigripalpus, and Cx. pipiens. To that list we have added An. punctipennis,An. stephensi,Cx. pipiens quinquefasciatus, and Cs. impatiens for a total of nine species and four genera of mosquitoes.

Based on the temporal proximity between MPM2 formation and adult emergence, MPM2 formation possibly is stimulated by the midgut epithelium that occurs at that time (Romoser 1974). In some of the species studied as adults, although MPM2 was found in the 0- to 4-h group, its frequency of occurrence increased substantially in the later groups. In addition, we often observed gas bubbles within the confines of MPM2 as reported earlier (Romoser 1974). These bubbles could be gas trapped within forming MPM2 or gas that seeps into the space between the relatively loose MPM2 and the MPM1. If MPM2 formation is distention-induced, it is probably the same mechanism that operates when PM is induced to form by the distention associated with the anal injection of gas or a blood meal (Freyvogel and Jaquet 1965). In the cases where membrane interpreted as MPM2 formed before midgut distention, this membrane could represent a second membrane similar to MPM1, although in no cases were three membranes observed around the meconium.

At this point, it is unclear whether or not gut expansion is the stimulus for MPM2 formation and more study is necessary. Because MPM2 is formed by a fully developed adult midgut and often has an associated plug similar to the PM plug that forms around a blood meal (Richardson and Romoser 1972, Romoser and Cody 1975), MPM2 is probably the same or very similar to the PM that forms around a blood meal. Ultrastructural studies would be useful in this regard.

In regard to the functions of the MPMs, at least two hypotheses have been proposed (Moncayo and Romoser 1994). First, MPMs may serve to wall off and contain potentially harmful microorganisms present in the gut lumen of the generalist feeding larvae. Second, MPMs may protect the developing adult midgut epithelial cells from hydrolytic enzymes associated with meconial degeneration. It is possible that both hypotheses are correct. However, the fact that MPM1 is not always formed in Ae. aegypti is not consistent with the hydrolytic enzyme hypothesis.

Acknowledgements

The authors acknowledge the dedicated assistance of Kristine Oswald. The research reported here has been supported through grants from the Ohio University Research Committee.

Author notes

2

Center for Tropical Disease and Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555–0609.

3

Department of Entomology, U.S. Army Medical Component (USAMC), Armed Forces Institute of Medical Research (AFRIMS), 315/6 Rajvithi Road, Bangkok 10400, Thailand.