The association between non-biting midges and ... - Wiley Online Library

Environmental Microbiology (2008) 10(12), 3193–3200

doi:10.1111/j.1462-2920.2008.01714.x

Minireview The association between non-biting midges and Vibrio cholerae Meir Broza,1 Hanan Gancz2 and Yechezkel Kashi3* 1 Department of Biology, Faculty of Science and Science Education, University of Haifa, Oranim, Tivon 36006, Israel. 2 Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA. 3 Department of Biotechnology and Food Engineering, The Technion – Israel Institute of Technology, Haifa 29000, Israel. Summary Vibrio cholerae is a natural inhabitant of aquatic ecosystems, yet its interactions within this habitat are poorly understood. Here we describe the current knowledge on the interaction of V. cholerae with one group of co-inhabitants, the chironomids. Chironomids, non-biting midges (Chironomidae, Diptera), are an abundant macroinvertebrate group encountered in freshwater aquatic habitats. As holometabolous insects, chironomids start life when their larvae hatch from eggs laid at the water/air interface; through various feeding strategies, the larvae grow and pupate to become short-lived, non-feeding, adult flying insects. The discovery of the connection between V. cholerae and chironomids was accidental. While working with Chironomus transavaalensis, we observed the disintegration of its egg masses and searched for a possible microbial agent. We identified V. cholerae as the primary cause of this phenomenon. Haemagglutinin/protease, a secreted extracellular enzyme, degraded the gelatinous matrix surrounding the eggs, enabling bacterial growth. Observation of chironomids in relation to V. cholerae continuously for 7 years in various types of water bodies in Israel, India, and Africa revealed that environmental V. cholerae adhere to egg-mass surfaces of various Chironomini (‘bloodworms’). The flying adults’ potential to

Received 6 February, 2008; accepted 22 June, 2008. *For correspondence. E-mail [email protected]; Tel. (+972) 4 8293074; Fax (+972) 4 8293399.

serve as mechanical vectors of V. cholerae from one water body to another was established. This, in turn, suggested that these insects play a role in the ecology of V. cholerae and possibly take part in the dissemination of the pathogenic serogroups during, and especially between, epidemics.

Introduction An accidental discovery in a pond of rehabilitated water During the early 1990s, chironomids became pestiferous insects in various water systems in Israel, including potable water systems (Broza et al., 1998; 2000; 2003). To find an acceptable control method for Chironomus transavaalensis (a major species), large numbers of egg masses were repeatedly collected from wastestabilization pond (Fig. 1) and brought to the lab. After one of these collections, egg masses that were left overnight in the laboratory were found to have ‘disappeared’ the next morning. Thousands of single eggs, released from the gelatinous matrix, were observed on the bottom of the container, most of them unhatched. In an effort to explain this occurrence, we isolated a bacterium that indeed released heat-labile factors causing the gelatinous matrix of Chironomini eggs to degrade (Fig. 2). This bacterium was identified as Vibrio cholerae and was serotyped as serogroup O9 (Broza and Halpern, 2001). Serogroup O9 belongs to the non-O1/O139 ‘environmental’ strains. Vibrio cholerae is an native microorganism of aquatic ecosystems, but the details of its interactions with other inhabitants of its natural habitat remain mostly unknown. The above-described finding and subsequent work on the relationship between these bacteria and the chironomids shed light on this generally overlooked aspect of V. cholerae’s survival as a free-living organism in the environment.

Vibrio cholerae: an aquatic bacterium and human pathogen More than 200 serogroups of V. cholerae are known (Shimada et al., 1994; E. Arakawa, unpublished);

© 2008 The Authors Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd

3194 M. Broza, H. Gancz and Y. Kashi A

Fig. 1. Trapping of chironomid egg masses (CEM) on styrofoam board. A. Styrofoam board (25 ¥ 25 cm) floating on the surface of a rehabilitated water pond. An evening breeze (in the picture – from left to right) forced the females to lay egg masses on the side protected from the wind. Hundreds of gelatinous CEM are glued to each other and covered by dead females (post-oviposition). B. Egg masses of two species of chiromonids attached to the styrofoam board. The bar represents 7 mm (photo: Y.Y. Broza).

B

however, only serogroups O1 and O139 have been associated with severe cholera pandemics (Kaper et al., 1995; Colwell, 1996; Harvell et al., 1999). The fatal effects of the disease are attributed to secretion of the cholera toxin (CT) produced by these serogroups (Davis and Waldor, 2003). However, CT is secreted by bacteria which belong to the other serogroups as well, and the existence of homologous toxins has been demonstrated (Jiang et al., 2003; Maiti et al., 2006). All other serogroups are referred to as ‘environmental strains’, although many of them may cause cholera with mild diarrhoea, associated with locally confined outbreaks. Nevertheless, there is some concern that new pathogenic, pandemic-causing strains will emerge from the environment as did the pathogenic serogroup O139 in 1992 (Faruque and Nair, 2002). As it became clear that V. cholerae infection is not chronic in humans, it was assumed that during the interepidemic period, the bacteria exist as natural inhabitants of aquatic ecosystems. Nevertheless, the mode of survival and means of distribution of environmental and pathogenic strains of V. cholerae remained to be clarified. Today, mounting evidence clearly points to the adaptability of V. cholerae to life as a pathogen of aquatic insects (Kirn et al., 2005; Meibom et al., 2005; Blokesch and Schoolnik, 2007), similar to other environmental members of the family Vibrionaceae, which are pathogens of fish and prawns, among others.

Chironomids The Chironomidae (Armitage et al., 1995) are a group of flies (Diptera) of the suborder Nematocera. Their common names are chironomids, non-biting midges, or ‘blind mosquitoes’ as adults, and ‘bloodworms’ as larvae. Chironomids are closely related to mosquitoes (Culicidae) and biting midges (Ceratopogonidae), but the adult female does not bite humans, or feed at all for that matter, and the same holds true for males. Chironomidae undergo complete metamorphosis in four life stages (Fig. 3): eggs, larvae, pupae (all of which are aquatic) and adults (imago) that emerge into the air. Chironomidae are usually the most abundant macroinvertebrate group encountered in most freshwater aquatic habitats, in terms of both number of species, number of individuals as well as biomass turnover rate (Benke, 1998). Chironomids are distributed worldwide, and as such are the only free-living holometabolous (i.e. having a four-stage life cycle) insects with true global distribution. Females of Chironomus sp. lay egg masses at the water’s edge. Each egg mass (Fig. 2) contains c. 400–2000 eggs (Hinton, 1981) embedded in a thick, gelatinous matrix (Nolte, 1993). The presence of several thousand egg masses at one site is not unusual, and in extreme cases, layers that are several centimetres thick may form along the water’s edge (Nolte, 1993; Broza et al., 2000; 2003).

Fig. 2. Chironomus egg mass. A. Healthy eggs, original magnification (40¥). Eggs are arranged in a row, folded into loops to form a spiral and embedded in a thick layer of gelatinous cylinder. Enlargement reveals the string-shaped mass and the gelatinous edge (egg mass size 20 ¥ 5 mm; egg size 250 ¥ 80 mm). B. Twelve hours after exposure to V. cholerae in minimal medium, the eggs protrude from the partially consumed gelatinous matrix. The bar in (A) represents 1 mm and in (B) represents 250 mm.

© 2008 The Authors Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 10, 3193–3200

Vibrio cholerae and chironomids 3195

Fig. 3. Life cycle of a chironomid. Chironomidae undergo complete metamorphosis in four life stages: eggs, larvae, pupae (all of which are aquatic) and adults (imago) that emerge into the air. (D) shows the skin of the pupa, the exuvia. Note the respiratory organ on the top (and see text, section Chironomid exuviae: a shell covered with V. cholerae). The bar in (A) and (D) represents 1 mm and in (B) and (C) 2 mm.

Chironomid egg masses as natural reservoir of V. cholerae Although V. cholerae had been isolated from various aquatic organisms, such as phytoplankton, oysters and crabs (Islam et al., 1996), there was no indication of their propagation on these organisms. Colwell and collaborators (Huq et al., 1983; Colwell, 1996; Lipp et al., 2002; Rawlings et al., 2007) showed an association between pathogenic V. cholerae and zooplankton, notably copepods. It is suggested here that both copepods and chironomids interact with V. cholerae. This interaction, however, may take place in different aquatic environments, each dominated by one of these arthropods, or may even occur in a complementary fashion on a spatial-temporal axis in the same water ecosystem. Egg masses of chironomids harbouring V. cholerae were collected, continuously, in 12 different freshwater bodies in Israel, and in several locales throughout India (Halpern et al., 2004; Broza et al., 2005; N. Raz, Y.Y. Broza, Y. Danin-Poleg, and Y. Kashi, in preparation). Fiftyone environmental serogroups of V. cholerae (out of 210 presently known antigens) were identified from those samples. Inter-strain variability was elucidated by SSR (simple sequence repeat) typing (e.g. 118 environmental strains isolated with 60% SSR types), which emphasized the overwhelming diversity of environmental V. cholerae in chironomid egg masses (Danin-Poleg et al., 2007; N. Raz, Y.Y. Broza, Y. Danin-Poleg, and Y. Kashi, in preparation). The enzyme responsible for degradation of the chironomid egg masses’ gelatinous matrix was purified from V. cholerae and identified as haemagglutinin protease (HA/P) (Halpern et al., 2003). This enzyme has been found in all V. cholerae strains examined. Although the

role of V. cholerae HA/P in human pathogenesis has not yet been elucidated (Finkelstein et al., 1992), it is clear that Vibrio needs HA/P in order to utilize the egg masses as a food source, facilitating its environmental survival. This ability of HA/P indicates an environmental role for one of the most abundant secreted proteins of V. cholerae and other aquatic bacteria, including Aeromonas hydrophila and Pseudomonas aeruginosa (Halpern et al., 2003), and strengthens Sylwen’s (1977) hypothesis that the human–cholera association has only been established in recent centuries, a hypothesis strongly asserted by Blow and colleagues (2005). Interestingly, Blow and co-workers (in the laboratory of P.I. Watnick) succeeded in simulating a cholera-like disease in Drosophila, and went on to state that: ‘We present the provocative hypothesis that the pathogenic program of V. cholerae may have evolved for an arthropod rather than for us.’ Recent work with Caenorhabditis elegans (Vaitkevicius et al., 2006) as well as the aquatic Protists (Abd et al., 2005; 2007) further supports this hypothesis. New information (Halpern et al., 2007) suggests that chironomid egg masses harbour V. cholerae in both viable and VBNC (viable but not culturable) forms: the culturable bacteria ranged from 6 to 36 per egg mass, less than 0.5% of the V. cholerae present. These findings strengthened the definition of chironomids as a ‘natural reservoir’ for this bacterium. Attachment of the chironomid egg mass to a substrate at the water’s surface appears to expose the mass as a nutritious food source for omnivorous and planktonic organisms in that water body, be they parasites, predators or hitchhikers. Aside from V. cholerae, 20 different bacteria, belonging to eight genera, were isolated in a year-long survey of chironomid egg masses (Halpern et al., 2007), supporting the possible role of chironomid egg masses and chironomid adults as,


3196 M. Broza, H. Gancz and Y. Kashi respectively, environmental reservoirs for, and carriers of V. cholerae and other associated bacteria. We therefore suggest considering V. cholerae and other related species as natural parasites of chironomids. Anecdotal findings or a new perspective? Vibrio cholerae survival in the environment Although sporadic and sometimes anecdotal reports on the interactions of V. cholerae with its aquatic environment have been around for over three decades, it is only since the pioneering – and ongoing – work of Rawlings and colleagues (2007) that several investigators have attempted to find an environmental function for V. cholerae’s arsenal of virulence factors. The effectiveness of these virulence factors against flies (Blow et al., 2005) and nematodes (Vaitkevicius et al., 2006), as well as V. cholerae’s ability to sense and respond to chitin (Kirn et al., 2005; Meibom et al., 2005; Blokesch and Schoolnik, 2007), indicates these bacteria’s adaptation to arthropod-derived nutrients. Although nematodes do not belong to the arthropods, the two phyla were recently paired based on the production of a cuticular exoskeleton and 18S ribosomal (rDNA) sequence data (see Giriber et al., 2000). The current view strongly supports incorporating nematodes and arthropods into a new clad – Ecdysozoa (molting animals), as both produce the hormone ecdyson and molt external cuticle, and both have been suggested to be associated with V. cholerae involvement in chitin degradation and pathogenesis. Is the spread of cholera related to mechanical aerial transmittance of V. cholerae by chironomid adults? Vibrio cholerae can survive in both freshwater and marine habitats (Colwell, 1996). Sea-borne currents and seaborne transportation represent prevailing modes for dissemination of the disease worldwide. Land transportation, festivals and pilgrimages to holy cities have also been suggested to contribute to its spread. However, the occurrence of simultaneous cholera outbreaks in distant locales deep within continental land masses, their immediate cause and means of dissemination, remain a mystery. We have presented evidence that flying, non-biting midges, collected in the air, carry environmental (non-01/ non-0139) V. cholerae attached to their inter-segmental membranes (Fig. 4) (Broza et al., 2005). Supported by experiments in a controlled environment, we were able to demonstrate that this potential holds true for pathogenic strains of V. cholerae as well. We suggested that chironomids can serve as a mechanical vector transporting V. cholerae over land from one water body to the next. In the laboratory, the vector potential of adult flying chi-

Fig. 4. Green fluorescent protein (GFP)-tagged Vibrio cholerae serotype O9 on an adult chironomid. Two abdominal segments are shown. Adults that emerged from the water contained GFP-tagged bacteria (GFP-encoding plasmid for 24 h), original magnification 100¥. The bar represents 0.5 mm.

romonids was explored using V. cholerae tagged with a green fluorescent protein (GFP)-encoding plasmid. The GFP-tagged bacteria were introduced into a laboratory flask with fourth-instar chironomid larvae. Adults subsequently emerged from the pupae into the air. Vibrio cholerae were readily detected on those adults. Some of the adult females laid egg masses in the water of an external container and GFP-tagged V. cholerae were isolated from that water, indicating transport by the contaminated chironomid adults. Vibrio cholerae O1 and O139 were indistinguishable in these experiments from V. cholerae O9, with respect to their ability to be carried by the chironomid adults. The vector potential of adult flying chironomids was further demonstrated in a field experiment testing the efficacy of different-mesh-size nets in preventing V. cholerae colonization of artificial water bodies. The large-size mesh proved to be ineffective at preventing V. cholerae colonization of a water body, while the small-mesh-size net, which adequately prevented chironomid access, completely eliminated the detection of V. cholerae in the water above which it was spread. Simultaneously, V. cholerae was detected on flying adult chironomids and from egg masses laid in the accessible pools. We were not successful in isolating V. cholerae from other flying insects that were caught alongside the chironomids. Adults of non-biting midges (Chironomidae and Chaoboridae) usually emerge and create swarms for mating that may include milliards of adults. From such a swarm observed in Kenya (Fig. 5) V. cholerae O2 was isolated (Broza et al., 2005). Based on these findings, we presented a hypothesis that aerial transfer by flying insects may play a role in the continental and intercontinental dissemination of cholera (Broza et al., 2005). There have been no systematic observations of longdistance movement of adult midges, as ‘aero-plankton’. A screening of the general entomological literature


Vibrio cholerae and chironomids 3197 Paz and Broza (2007). It was linked with unusual atmospheric conditions that differed significantly from the mean of the previous 50-year period (1940-1990). Cholera epidemics usually start on the eastern shore of India (The Bay of Bengal) and then proceed west and north-west (G.B. Nair, pers. comm., see also discussion in Paz and Broza, 2007). They are correlated with south-easterly winds during the beginning and the end of summer’s rainy monsoon. How can winds influence water-borne bacteria? One possibility is via drifting swarms of flying insects (Fig. 6). Chironomid exuviae: a shell covered with V. cholerae Fig. 5. Lake Victoria, Kenya. A huge swarm of non-biting midges (of sister group, family Chaoboridae) was followed by the first author for at least 12 km, moving downwind, northward. Hundreds of them were caught by light trap on the shore at Mbita Point experimental station and V. cholerae serotype O2 was isolated from those adults (photo taken in 2001 by Hana Nadel).

revealed accidental data on trapping adult non-biting midges high in the air or on ships, well into the open ocean, far from the seashore where they could breed (Table 1). Reynolds and colleagues (1999), who used aerial netting at a height of 200 m to observe the migration of rice pests from China into the Ganga Valley, identified chironomid adults as part of the trapped insects and in one case, about 50% of the identified trapped insects were adult chironomids. Recently, Paz and Broza (2007) suggested a linkage between dominant wind direction and spread of pandemic cholera, in both Africa (Fig. 6) and India. The relationship between cholera epidemics and climatic events has been previously proposed (Lipp et al., 2002), but that research focused on linkages to the main climatic factors, such as the amount of precipitation, air temperature and sea surface temperature. No correlation with wind was mentioned. The direction of the rapid spread of the new pathogenic isolate (O139) in the Indian subcontinent in 1992-1993 (Ramamurthy et al., 1993) was analysed by

The previous sections dealt with two of the chironomids’ four life-cycle stages: egg masses and adults. Vibrio cholerae, however, have also been isolated from the other two life stages of the midges, the bottom tube-dwelling larvae and the active pupae. Pupation takes place inside the larval tube; the active pupa then climbs up to the water surface, hanging itself, while the air-filled respiratory organ keeps the front of the pupa slightly above water, enabling the fresh adult to emerge and fly straight into the air, leaving the pupa skin, called exuvia, to float on the water’s surface (Fig. 3D). These exuviae constitute easily accessible evidence of the chironomids’ presence and may adequately represent the V. cholerae strains that find their way to the air. The chironomid exuvia is a unique structure, which includes a floating net-like cuticle made of chitin. Exuviae are species-specific (see Langton and Visser, 2006), and ecologists use them to sample and monitor adult populations in streams (Wilson and McGill, 1977; Ruse and Wilson, 1994). We assumed that the exuviae would serve as a good monitoring unit for V. cholerae, as the bacteria readily attach to them (Fig. 7, H. Gancz, M. Broza and Y. Kashi, unpublished). Indeed, we have recently used exuviae to monitor the occurrence of V. cholerae in streams and other water bodies in central and southern

Table 1. Reports of trapping air-borne chironomids over oceans on ships (S) and oil platforms (R), or high in the air, with aircraft (A) or kytoon (K). Nearest dryland location

Year

Offshore (km)

Brazil Uruguay Okinawa Mexico Louisiana Galapagos Louisiana Australia Australia W. Bengal

1964 1964 1964 1968 1973 1993 1935 1970 1971 1996

(S) 30–90 (S) 15–25 (S) ~60 (S) 155 (R) 32–105 (S) 2–100

Height (mASL)

No. of specimens

References

(A) (A) (A) (K)

63 287 98 5 564 17 ~1000 105 50 332

Clagg (1966) Clagg (1966) Harrel and Holzapfel (1966) Holzapfel and Perk (1969) Spark (1986) Peck (1994) Glick (1939) White (1970) White (1974) Reynolds et al. (1999)

60–1500 ~600 ~300 150

mASL, metres above sea level. All trappings were part of general entomological surveys, not especially devoted to chironomids.


3198 M. Broza, H. Gancz and Y. Kashi

Fig. 6. Cholera outbreaks in Africa during 2005-2006, correlated with mean wind direction. (From Paz and Broza, 2007.) SLP stands for sea level pressure.

India (N. Raz, Y.Y. Broza, Y. Danin-Poleg, and Y. Kashi, in preparation). We expect that in the future, exuvia monitoring for the presence of V. cholerae will become a more common technique due to the simplicity of their collection.

Concluding remarks The occurrence of cholera, particularly with respect to its wide pandemic characteristics, is still a mysterious


Vibrio cholerae and chironomids 3199 Fig. 7. GFP-tagged Vibrio cholerae, attached to the respiratory organ of the chironomid pupa (and exuvia). Examination was performed under a microscope with fluorescent light (right) and visible light (left). The red particles seen next to the green shiny bacteria in the right panel are the unicellular alga, Navicula (Diatomae), original magnification 200¥. The bar represents 0.15 mm. (Figs 4 and 7 by Hanan Gancz.)

phenomenon. But even more astonishing are the abrupt disappearance of the disease and the fact that there are no chronic cases in humans. Thus, for many years, researchers have attempted to find where the bacteria reside during the inter-epidemic periods. Arthropods of various taxa (e.g. copepods) appear to meet V. cholera in the water ecosystem and may serve as a nutritional source for this bacterium. The reported observations connecting V. cholerae to chironomids support our hypothesis that chironomid egg masses serve as a natural reservoir for V. cholerae. The massive volume of egg masses appearing at the edge of freshwater bodies coincides with the area from which drinking water is traditionally drawn, and where people bathe. In addition, adult chironomids have been shown to carry environmental V. cholerae strains attached to their bodies, thereby transferring the bacteria between nearby bodies of water. How far this route extends, and whether it is a major route of cholera dissemination, remain open questions. Acknowledgements This research was supported by the Israel Science Foundation Grant 1005697, The Grand Water Research Institute, Technion and The Israeli Water Commission.

References Abd, H., Weintraub, A., and Sandstrom, G. (2005) Intracellular survival and replication of Vibrio cholerae O139 in aquatic free-living amoebae. Environ Microbiol 7: 1003– 1008. Abd, H., Saeed, A., Weintraub, A., Nair, G.B., and Sandstrom, G. (2007) Vibrio cholerae O1 strains are facultative intracellular bacteria, able to survive and multiply symbiotically inside the aquatic free-living amoeba Acanthamoeba castellanii. FEMS Microbiol Ecol 60: 33–39. Armitage, P., Cranston, P.S., and Pinder, C.V. (1995) The Chironomidae: The Biology and Ecology of Non-biting Midges. London, UK: Chapman & Hall. Benke, A.C. (1998) Production dynamics of riverine chironomids: extremely high biomass turnover rates of primary consumers. Ecology 79: 899–910.

Blokesch, M., and Schoolnik, G.K. (2007) Serogroup conversion of Vibrio cholerae in aquatic reservoirs. PLoS Pathog 3: e81. doi:10.1371/journal.ppat.0030081. Blow, N.S., Salomon, R.N., Garrity, K., Revellaud, I., Kopin, A., Jackson, F.R. et al. (2005) Vibrio cholerae infection of Drosophila melanogaster mimics the human disease cholera. PLoS Pathogens 1: 92–98. Broza, M., and Halpern, M. (2001) Chironomids egg masses and Vibrio cholerae. Nature 412: 40. Broza, M., Halpern, M., Teltsch, B., Porat, R., and Gasith, A. (1998) Shock chloramination: a potential treatment for Chironomidae (Diptera) larvae nuisance abatement in water supply systems. J Econ Entomol 91: 834–840. Broza, M., Halpern, M., and Inbar, M. (2000) Non-biting midges (Diptera; Chironomidae) in waste stabilization ponds: an intensifying nuisance in Israel. Water Sci Technol 42: 71–74. Broza, M., Halpern, M., Gahanma, L., and Inbar, M. (2003) Nuisance chironomids in waste stabilization ponds: monitoring and action threshold assessment, based on public complaints. J Vector Ecol 28: 31–36. Broza, M., Gancz, H., Halpern, M., and Kashi, Y. (2005) Adult non-biting midges: possible windborne carriers of Vibrio cholerae non-O1 non-O139. Environ Microbiol 7: 576–585. Clagg, H.B. (1966) Trapping airborne insects in the Atlantic– Antarctic area. Pac Insects 8: 33–42. Colwell, R.R. (1996) Global climate and infectious disease: the cholera paradigm. Science 274: 2025–2031. Danin-Poleg, Y., Cohen, L.A., Gancz, H., Broza, Y.Y., Goldshmidt, H., Malul, E., et al. (2007) Vibrio cholerae strain typing and phylogeny study based on simple sequence repeats (SSR). J Clin Microbiol 45: 736–746. Davis, B.M., and Waldor, M.K. (2003) Filamentous phages linked to virulence of Vibrio cholerae. Curr Opin Microbiol 6: 35–42. Faruque, S.M., and Nair, G.B. (2002) Molecular ecology of toxigenic Vibrio cholerae. Microbiol Immunol 46: 59–66. Finkelstein, R.A., Bosman-Finkelstein, M., Chang, Y., and Hass, C.C. (1992) Vibrio cholerae hemagglutinin/protease, colonial variation, virulence, and detachment. Infect Immun 60: 472–478. Giriber, G., Distel, D.L., Polz, M., Sterrer, W., and Wheeler, W.C. (2000) Triploblastic relationships with emphasis on the acoelomates and the position of Gnathostomulida, Cycliophora, Plathelminthes, and Chaetognatha: a combined approach of 18S rDNA sequences and morphology. Syst Biol 49: 539–562.


3200 M. Broza, H. Gancz and Y. Kashi Glick, P.A. (1939) The distribution of insects, spiders and mites in the air. US Dep Agric Techn Bull 673: 1–151. Halpern, M., Gancz, H., Broza, M., and Kashi, Y. (2003) Vibrio cholerae hemagglutinin/protease degrades chironomid egg masses. Appl Environ Microbiol 67: 4200–4204. Halpern, M., Broza, Y.B., Mittler, S., Arakawa, E., and Broza, M. (2004) Chironomid egg masses as a natural reservoir of Vibrio cholerae non-O1 and non-O139 in freshwater habitats. Microb Ecol 47: 341–349. Halpern, M., Landsberg, O., Raats, D., and Rosenberg, E. (2007) Culturable and VBNC Vibrio cholerae: interactions with chironomid egg masses and their bacterial population. Microb Ecol 53: 285–293. Harrell, J.C., and Holzapfel, E. (1966) Trapping airborne insects on ships in the Pacific, Part 6. Pac Insects 8: 33–42. Harvell, C.D., Kimm, K., Burkholderm, J.M., Colwell, R.R., Epstein, P.R., Grimes, D.J., et al. (1999) Emerging marine diseases – climate links and anthropogenic factors. Science 285: 1505–1510. Hinton, H.E. (1981) Biology of Insect Eggs. Oxford, UK: Pergamon Press. Holzapfel, E.P., and Perkins, C. (1969) Trapping of airborne insects on ships in the Pacific, Part 7. Pac Insects 11: 455–476. Huq, A., Small, E.B., West, P.A., Rahman, R., and Colwell, R.R. (1983) Ecology of V. cholerae with special reference to planktonic crustacean copepods. Appl Environ Microbiol 45: 275–283. Islam, M.S., Drasar, B.S., and Sack, R.B. (1996) Ecology of Vibrio cholerae. In Cholera and the Ecology of Vibrio cholerae. Drasar, B.S., and Forrest, B.D. (eds). London, UK: Chapman & Hall, pp. 187–227. Jiang, S., Chu, W., and Fu, W. (2003) Prevalence of cholera toxin genes (ctxA and zot) among non-O1/O139 Vibrio cholerae strains from Newport Bay, California. Appl Environ Microbiol 69: 7541–7544. Kaper, J.B., Morris, J.G., and Levine, M.M. (1995) Cholera. Clin Microbiol Rev 8: 48–86. Kirn, T.J., Jude, B.A., and Taylor, R.K. (2005) A colonization factor links Vibrio cholerae environmental survival and human infection. Nature 438: 863–866. Langton, P.H., and Visser, H. (2006) Chironomidae Exuvia – A Key to Pupal Exuviae of the West Palaearctic Region. Hybrid CD. Amsterdam, the Netherlands: ETI Bioinformatics. Lipp, E.K., Huq, A., and Colwell, R.R. (2002) Effects of global climate on infectious disease: the cholera model. Clin Microbiol Rev 15: 757–770. Maiti, D., Das, B., Saha, A., Nandy, R.K., Nair, G.B., and Bhadra, R.K. (2006) Genetic organization of pre-CTX and CTX prophages in the genome of an environmental

Vibrio cholerae non-O1, non-O139 strain. Microbiology 152: 3633–3641. Meibom, K.L., Blokesch, M., Dolganov, N.A., Wu, C.Y., and Schoolnik, G.K. (2005) Chitin induces natural competence in Vibrio cholerae. Science 310: 1824–1827. Nolte, U. (1993) Egg masses of Chironomidae (Diptera). A review, including new observations and a preliminary key. Entomol Scand Suppl 43: 5–75. Paz, S., and Broza, M. (2007) Wind direction and its linkage with Vibrio cholerae dissemination. Environ Health Perspect 115: 195–200. Peck, S.B. (1994) Aerial dispersal of insects between and to islands in the Galapagos Archipelago, Ecuador. Ann Entomol Soc Am 87: 218–224. Ramamurthy, T.S., Sharma, G.R., Bhattacharya, S.K., Nair, G.B., Shimada, T., Takeda, T., et al. (1993) Emergence of a novel strain of Vibrio cholerae with epidemic potential in southern and eastern India. Lancet 341: 703–704. Rawlings, T.K., Ruiz, G.M., and Colwell, R.R. (2007) Association of Vibrio cholerae O1 El Tor and O139 Bengal with the Copepods Acartia tonsa and Eurytemora affinis. Appl Environ Microbiol 73: 7926–7933. Reynolds, D.R., Mukhopadhyay, S., Riley, J.R., Das, B.K., Nath, P.S., and Mandal, S.K. (1999) Seasonal variation in windborne movement of insect pests over northeast India. Int J Pest Manag 45: 195–205. Ruse, L.P., and Wilson, R.S. (1994) Long term assessment of water and sediment quality of the River Thames using chironomid pupal skin. In Chironomids: From Genes to Ecosystems. Cranston, P. (ed.). East Melbourne, Australia: CSIRO Publications, pp. 113–123. Selwyn, S. (1977) Cholera old and new. Proceeding of the Royal Society Medicine 70: 301. Shimada, T., Arakawa, E., Itoh, K., Okitsu, T., Matsushima, A., Asai, Y., et al. (1994) Extended serotyping scheme for Vibrio cholerae. Curr Microbiol 28: 175–178. Spark, A.N., Jackson, R.D., and Carpenter, J.E. (1986) Insects captured in light traps in the Gulf of Mexico. Ann Entomol Soc Am 79: 132–139. Vaitkevicius, K., Lindmark, B., Ou, G., Song, T., Toma, C., Iwanaga, M., et al. (2006) Vibrio cholerae protease needed for killing of Caenorhabditis elegans has a role in protection from natural predator grazing. Proc Natl Acad Sci USA 103: 9280–9285. White, T.C.R. (1970) Airborne arthropods collected in South Australia with a drogue-net towed by a light aircraft. Pac Insects 12: 251–259. White, T.C.R. (1974) Arthropods occurring in the air over South Australia. Pac Insects 16: 1–10. Wilson, R.S., and McGill, J.D. (1977) A new method of monitoring water quality in a stream receiving sewage effluent, using chironomid pupal exuviaae. Water Res 11: 659–662.
