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Technique for mass rearing of Indian Chironomus species

  • Savitribai Phule Pune University Pune India
NATH& GODBOLE:187-193 Studia dipterologica 5 (1998) Heft 2
Technique for mass rearing of Indian Chironomus species
(Diptera, Nematocera, Chironomidae)
[Eine Technik zur Massenzucht von indischen Chironomus-Arten
(Diptera, Nematocera, Chironomidae)]
Bimalendu B. NATH and Narayan N. GODBOLE
Pune (India)
Key words
At present, no standard methodologies are available to colonize and rear tropical
midges of the family Chironomidae (Diptera). We present here a simple. inexpensive
laboratory method of mass rearing Indian Chirono/lllls, based on locally available
food. Morphometric analysis to monitor larval growth has been carried out to establish
suitability of the rearing methodology. The rearing technique described in this paper,
provides a continuously breeding population of three Indian species of Chirono/lllls.
mass rearing, Chironomidae, Chirono/lllls, India
Derzeit existiert keine Standartmethode, mit der tropische Zuckmlicken (Diptera,
Chironomidae) unter Laborbedingungen zu halten und zu vermehren sind. Es wird
eine einfache, wenig kostenintensive Labormethode zur Zucht yon Chirono/lllls-Ar-
ten der indischen Fauna vorgestellt, die auf derVerwendung eines einheimischen Zucht-
substrates beruht. Eine Uberwachung des Wachstums der Larven li\;)erMessungen der
Kopfkapselbreite wurde angewendet, urn die Brauchbarkeit der Zuchtmethode abzu-
sichern. Die hier geschilderte Methode wurde erfolgreich bei der kontinuierlichen
Haltung yon drei indischen Chirollollllls-Arten verwendet.
Massenzucht, Chironomidae, ChiroI:.0/llIIS,(ndien
Chironomid midges constitute a major biotic component in most of the freshwater eco-
system (OLIVER1971, PINDER1986). For decades, a great deal of research had been done
in cytogenetics and pollution biology using Chironomus as a system (MURRAY1980,
ARMITAGEet al. 1994). In the past, several workers described methods of rearing that
suited to their experimental conditions. It was however experienced that none of the
available methods were suitable either because of nonavailability of food components or
because of different conditions required by tropical midges. Hence a new method has
been evolved suitable for maintaining tropical Indian midges as a continuous culture
under laboratory con~itions.
A method for the propagation of Chironomus tentans FABRICIUS,1805, was reported by
SADLER(1935). However, alternative methods were utilized by subsequent workers for
rearing C. tentans (BENTIVEGNA& COOPER1993, FIRLING& KOBILKA1979, MEYERet al.
1983). BIEVER(1965) studied different species of Chironomus, namely C. attenuatus
WALKER, 1848; C. fulvipilus REMPEL,1939; C. monochromus VANDERWULP, 1874; C.
stigmaterus SAY, 1823; C. califomicus JOHANNSEN,1905 and described techniques for
rearing in the laboratory (reviewed by SINGH& MOORE1985). BIEVER(1965) also de-
scribed composition of food, conditions for oviposition and designing of rearing cages.
... Chironomus ramosus, a common Indian tropical midge, inhabits freshwater ecosystems and is adapted to thrive under adverse environmental conditions (Armitage et al., 1995;Nath and Godbole, 1998). These primitive groups of dipteran insects, show tolerance to wide range of biotic (hypoxia, heat) and abiotic (water pollutants) stress factors (Choi et al., 2000;Yoshimi et al., 2002;Callaghan et al., 2002;Haas et al., 2005;Hassell et al., 2006;Nowak et al., 2007;Vogt et al., 2007). ...
... Thirty days old fourth instar larvae of the tropical insect species of midge C. ramosus were reared in insectary of Bhabha Atomic Research Centre, Mumbai, India. The laboratory cultures were maintained using mass rearing technique (Nath and Godbole, 1998) at 25 ± 2°C with relative humidity 75 ± 10% and 14 h:10 h light and dark photoperiod cycle. ...
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A battery of enzymes from the eukaryotic antioxidant defense system was measured in salivary gland and in whole body extract of fourth instar larvae of Chironomus ramosus with an objective of finding any clue for the dipteran insect's capacity to tolerate heavy doses of ionizing radiation. Levels of activity of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GSH-Px) were quantified in 30 days old larvae exposed to LD 20 dose of gamma radiation. Compared to controls, activity of Cu,Zn-SOD increased 3 to 4 fold and catalase 2 fold in response to ionizing radiation stress, while activities of GR and GSH-Px enzymes were decreased. Among the other SOD isoenzymes, our results showed comparable levels of Mn-SOD and Cu,Zn-SOD activity in control and irradiated groups of larvae. The increase in levels of the Cu,Zn-SOD isoenzyme was also confirmed by Western blot and zymography supported by densitometric quantification. No evidence of Fe-SOD was found in C. ramosus larvae. These findings could help to explain the persistence of natural populations of Chiro-nomus in radioactively contaminated regions.
... Previously, several works on HSPs and heat shock inducible chromosomal loci were reported mainly in two European species, namely, Chironomus tentans Fabricius and Chironomus thummi (Kieffer) (Tanguay and Vincent 1981;Lezzi 1984;Carretero et al. 1991;Sass 1995;Martinez et al. 1997;Morcillo et al. 1997) and in one Indian species, Chironomus striatipennis Kieffer (Nath and Lakhotia 1989). In the present study, a tropical Indian species, Chironomus ramosus Chaudhuri, Das and Sublette, colonized in the laboratory (Nath and Godbole 1998) has been chosen to study behavioural response to heat shock. ...
... Larvae were originally obtained from an isofemale line of S/S wild type population following a breeding scheme described by Hardikar and Nath (2001). All developmental stages were maintained in the laboratory by using a mass rearing technique (Nath and Godbole 1998) and fourth instar larvae from the synchronous culture were used for the present study. ...
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All living cells respond to temperature stress through coordinated cellular, biochemical and molecular events known as “heat shock response” and its genetic basis has been found to be evolutionarily conserved. Despite marked advances in stress research, this ubiquitous heat shock response has never been analysed quantitatively at the whole organismal level using behavioural correlates. We have investigated behavioural response to heat shock in a tropical midge Chironomus ramosus Chaudhuri, Das and Sublette. The filter-feeding aquatic Chironomus larvae exhibit characteristic undulatory movement. This innate pattern of movement was taken as a behavioural parameter in the present study. We have developed a novel computer-aided image analysis tool “Chiro” for the quantification of behavioural responses to heat shock. Behavioural responses were quantified by recording the number of undulations performed by each larva per unit time at a given ambient temperature. Quantitative analysis of undulation frequency was carried out and this innate behavioural pattern was found to be modulated as a function of ambient temperature. Midge larvae are known to be bioindicators of aquatic environments. Therefore, the “Chiro” technique can be tested using other potential biomonitoring organisms obtained from natural aquatic habitats using undulatory motion as a behavioural parameter.
... Larvae collected from the Mula river were validated for taxonomic identification using morphological and cytotaxonomic keys [26,27] and reared in non-toxic plastic tubs (Ø = 35 cm) containing sterilized beach sand at the bottom (S2A Fig). The rearing procedure was described previously [28,29]. In brief, to initiate rearing,~100 late fourth instar larvae were transferred to a separate plastic tub. ...
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Vibrio cholerae causes the fatal cholera diarrhea. Chironomids (Diptera; Chironomidae) are abundant in freshwater aquatic habitats and estuaries and are natural reservoirs of V. cho-lerae. Until now, only the non-O1/O139 serogroups of V. cholerae were identified in chirono-mids. Here, we explored whether chironomids are natural reservoirs of V. cholerae O1/ O139 serogroups, which are associated with cholera endemics and pandemics. All four life stages of chironomids were sampled from two rivers, and a laboratory culture in Pune, India, and from a pond in Israel. In total, we analyzed 223 chironomid samples. The presence of V. cholerae O1/O139 serogroups was verified using molecular tools. Nine chironomid species were identified; of them, Chironomus circumdatus was the most abundant. The presence of V. cholerae serogroup O1 and the cholera toxin genes were detected in samples from all chironomid species. However, serogroup O139 was detected in only two chironomid species. Besides PCR to detect specific genes, a metagenomic analysis that was performed in three selected C. ramosus larvae, identified a list of virulence genes associated with V. cho-lerae. The findings provide evidence that chironomids are natural reservoirs of toxigenic V. cholerae O1/O139. Chironomid populations and V. cholerae show biannual peak patterns.
... Chironomus ramosus larva (acetocarmine staining) showing Balbiani ring regions identified using a previously published cytological reference map (Nath and Godbole, 1998). Fig. 4. Venn diagram constructed to illustrate the diversity and occurrence of silk proteins among the three well studied Chironomus species (C. ...
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Silk proteins secreted by salivary glands in the dipteran insect, Chironomus play a significant role as proteinaceous adhesives for construction of underwater housing nests by larvae. To date, only three Chironomus species, C. tentans Fabricius, C. pallidivittatus Malloch and C. riparius Meigen have been explored for characterization of their aquatic silk protein. Genes coding for silk proteins are located on specific chromosomal ‘puffs’ called Balbiani rings as well as non- Balbiani ring regions. Expression of these genes is closely regulated by developmental and hormonal alterations and environmental factors. Furthermore, pilot studies have postulated that silk proteins probably occur in diverse size classes grouped into large (~1000 kDa), intermediate (100-200 kDa) and small (≤100 kDa). Barring few preliminary reports that date back to the 1990s, the physical and bioproperties of silk from chironomid midges remain largely unknown, leading to paucity of updated information. This review was therefore aimed to compile existing literature database and to highlight the wide possibilities for commercialization of midge larval silk as a novel biopolymer.
... Isofemale lines of C. ramosus were maintained at 25 ± 2°C and 14 h light: 10 h darkness cycle. The larvae were supplemented with food as described by Nath and Godbole [33] . ...
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The larvae of the aquatic Chironomid midge (Diptera, Chironomidae) are known to survive in freshwater habitats exposed to stress especially that of hypoxic stress. Hence, a study of the effect of alterations in the levels of dissolved oxygen on the physiology of the larvae could help in establishing them as potential sensitive bio indicators for monitoring such freshwater habitats. A simple inexpensive and non-cumbersome experimental setup unlike commercially available sophisticated set-ups was designed and fabricated in the lab. The validation of this set-up was carried out and alterations in the hemoglobin levels in the larvae of the tropical midge species Chironomus ramosus exposed to hypoxic conditions were studied. It was observed that there was an increase in the level of hemoglobin on exposure to hypoxic conditions and the findings were further validated by studying the expression of hemoglobin gene at various time points during exposure to hypoxia. These findings suggest that the device designed could be used as an inexpensive and effective method for generating and maintaining the desired levels of dissolved oxygen as compared to several of the chemical and physical methods generally used. Further, this would be useful in studies to be done for the monitoring of hypoxia in freshwater habitats using Chironomus hemoglobin as a biomarker.
... Rearing of midge species in the insectary is essential for scientific investigations. Above all, midge species was maintained in the insectary following Nath and Godbole (1998), Habashy (2005), Nath et al. (2009) and Bhaduri et al. (2012). Midge larvae procured from the insectary were used as vehicle for delivering probiotics during experimental period. ...
Effects of probiotics encapsulated and ascorbic acid enriched chironomid midge larvae as live feed on growth and survival of 12 day old Clarias batrachus juveniles (average weight 0.4 ± 0.06 g) were observed with feeding experiment for four weeks. Presumptive probiotic strains Bacillus aryabhattai KP784311, B. flexus KR809411, and B. cereus KR809412 were considered for bioencapsulation of chironomid larvae, and ascorbic acid (vitamin C) was used further for bioenrichment of the same. Seven experimental diets, D0: non-bioencapsulated and non-bioenriched chironomid larvae (control), D1: chironomid larvae bioencapsulated with B. aryabhattai, D2: chironomid larvae bioencapsulated with B. flexus, D3: chironomid larvae bioencapsulated with B. cereus, D4: chironomid larvae bioencapsulated with mixture of three strains, D5: chironomid larvae non-bioencapsulated and bioenriched with ascorbic acid, and D6: chironomid larvae bioencapsulated with probiotics and bioenriched with ascorbic acid were fed to triplicate groups. The juveniles were fed ad libitum for four weeks and the result of D6 group showed significantly higher (P < 0.05) specific growth rate and survivability than other groups. Although the RNA content in the carcass was increased initially in all dietary groups, any significant change of the DNA was not noticed. The coefficient (r value) of 0.992 in experiment 1 and 0.993 in experiment 2 along with regression slopes suggested a positive correlation (0.01 level) between RNA: DNA ratio and specific growth rate.
... Mass rearing technique was used to grow cultures of Chironomus ramosus as described previously [17]. ...
Chironomus ramosus is known to tolerate high doses of gamma radiation exposure. Larvae of this insect possess more than 95% of hemoglobin (Hb) in its circulatory hemolymph. This is a comparative study to see effect of gamma radiation on Hb of Chironomus and humans, two evolutionarily diverse organisms one having extracellular and the other intracellular Hb respectively. Stability and integrity of Chironomus and human Hb to gamma radiation was compared using biophysical techniques like Dynamic Light Scattering (DLS), UV-visible spectroscopy, fluorescence spectrometry and CD spectroscopy after exposure of whole larvae, larval hemolymph, human peripheral blood, purified Chironomus and human Hb. Sequence- and structure-based Bioinformatics methods were used to analyze the sequence and structural similarities or differences in the heme pockets of respective Hbs. Resistivity of Chironomus Hb to gamma radiation is remarkably higher than human Hb. Human Hb exhibited loss of heme iron at a relatively low dose of gamma radiation exposure as compared to Chironomus Hb. Unlike human Hb, the heme pocket of Chironomus Hb is rich in aromatic amino acids. Higher hydophobicity around heme pocket confers stability of Chironomus Hb compared to human Hb. Previously reported gamma radiation tolerance of Chironomus can be largely attributed to its evolutionarily ancient form of extracellular Hb as evident from the present study.
... 73 ° 51 ′ 48.06 ′ ′ E) inhabited by the natural population of C. ramosus . Culture was maintained by mass rearing technique (Nath and Godbole 1998) at 25 Ϯ 2 ° C temperature, relative humidity of 50 Ϯ 10%, and a photoperiod of 14 h light and 10 h darkness in the insectary of Bhabha Atomic Research Centre (BARC), Mumbai. Th irty-day-old fourth instar larvae were put in a glass beaker containing tap water and exposed to a dose of 2200 Gy a lethal dose for 20% of the midge population (LD 20 ) determined by dosimetric studies for the determination of percent mortality of irradiated larvae followed by Probit analysis (Datkhile et al. 2009a) from a cobalt 60 Co source (dose rate 5.5 Gy/min, Gamma Cell 220, Atomic Energy of Canada Ltd, Ottawa, Canada). ...
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Chironomus ramosus is one of the recently reported radio-tolerant insects. Salivary gland cells of fourth instar larvae respond to ionizing radiations with increases in the levels of antioxidant enzymes and chaperone proteins. Here we made an attempt to study the state of nuclear DNA after exposure of larvae to a Lethal Dose for 20 percent of the population (LD20) of gamma radiation (2,200 Gy, at a dose rate 5.5 Gy/min). Genomic DNA preparations were subjected to competitive ELISA (Enzyme Linked Immuno-Sorbant Assay) for detection of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and dynamic light scattering (DLS) to monitor any radiation induced damage. Single salivary gland cells were subjected to alkaline single cell gel electrophoresis, (ASCGE) comet assay and pulsed field gel electrophoresis (PFGE) to check for DNA double strand breaks. Results from all four experimental procedures confirmed damage of nucleobases and fragmentation of nuclear DNA immediately after radiation. Forty-eight h after radiation exposure, modified 8-oxodG residues returned to basal level, homodispersity of genomic DNA reappeared, the length of comet tail regressed significantly (ASCGE) and PFGE pattern matched with that of high molecular weight unirradited DNA. Chironomus ramosus larvae showed control of DNA damage as observed over 48 h in post irradiation recovery which could be attributed to their ability to tolerate gamma radiation stress.
The effects of crude root extracts of Eichhornia crassipes (Marts) Solms on Chironomus ramosus Chaudhuri eggs and larvae are studied under laboratory conditions. Egg masses of C. ramosus that are subjected to varying concentrations of exudates (final concentrations 0.25–2.5%) show 100% viability. However, larvae reared further at these concentrations reveal a dose–response relationship, with an LC50 value of 1.33% and an LC90 value of 2.05%. A striking feature is the occurrence of supernumerary salivary glands in larvae (three glands per larva) reared in the 1%, 1.25%, 1.5% and 1.75% extracts. This deviates from the normal two glands per larva and may be explained by altered growth as a result of the putative bioactive compounds present in the hyacinth extracts. In nature, hyacinth and midges co-exist in many freshwater habitats, implying that hyacinth compounds present from natural leaching could be encountered by the larvae. These preliminary findings suggest that hyacinth can influence the larval development of midges, giving rise to long-term ecological implications.
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