Larval Food Analysis and Qualitative Determination of Exoenzyme-Producing Gut Bacteria in Adult Ceratopogonid Midges (Diptera)

INTRODUCTION

Family Ceratopogonidae is an enormously diverse group of small nematocerous midges, usually known as biting midges. In this family along with some other genera Culicoides Latreille [1], and Dasyhelea Kieffer [2], are important in having medico-veterinary and economic significance respectively. Many species of Culicoides are vectors of pathogenic protozoans, viruses, and filarial nematodes [3-5] causing Akabane, Blue Tongue disease, etc. [6, 7]. Some members of the genus Dasyhelea are pollinators of cocoa and rubber trees [8, 9]. In the case of female Culicoides, the adult stage is hematophagous, feeding on vertebrate blood, though there is report of natural sugar feeding habit also [10]. The larvae of some species of Culicoides are trophic generalists [11]. Adult stages of both the sexes belonging to the genus Dasyhelea rely on nutrition from the honeydew and nectar [12], larval stages of this genus are primarily herbivorous actively feeding upon the plant and animal debris [13-15]. However, there is also evidence of carnivorous feeding habit of D. pseudoincisurata Waugh and Wirth, [16, 17].

Insects harbor a broad variety of microorganisms in their gut which help in numerous physiological functions [18]. There is also role of autochthonous bacteria in production of digestive enzymes for plant-derived polymers [19]. Digestion of lipid and protein may also be contributed by these microorganisms [20].

This article aims to evaluate the composition of food materials ingested by the larvae during active feeding stage. So far, no investigation has been made for qualitative determination of autochthonous enzyme-producing gut bacteria of adults Culicoides and Dasyhelea which we have also done in this study.

MATERIALS AND METHODS

Analysis of larval food (Figures 1a-1c)

A site (Site: India, West Bengal, Nadia, Krishnanagar) was selected for the collection of food material. Rotting banana stems were found at the collection site and the presence of flying adults confirmed the breeding habitat. Two types (Sample 1 and Sample 2) of the rotting stems were cut out with the help of a fine knife, bagged in 45×40 cm clear plastic packets, and brought to the laboratory keeping the packets airtight. The samples were kept in a 30 cm × 25 cm × 4 cm plastic tray with a little amount of water just to make the sample moistened. A small amount of sample was taken and examined carefully for the presence of any larva. Some larvae were observed underneath the Olympus SZX 16 microscope (Japan) and found consuming their habitat substrata which are the upper thin membranous leaflets of the stem in both the samples. Few larvae were isolated and reared up to adult emergence. Upon the adult eclosion, the midges were identified under Carl Zeiss Stemi 2000 Stereozoom microscope (Germany) and confirmed their belonging to the genera Culicoides and Dasyhelea. Further species-level identification was confirmed after mounting in glass slides and observing under a Wild Leitz GMBH Trinocular microscope (Portugal). The upper thin membranous layer of both samples was isolated carefully by forceps, washed thoroughly in tap water, and sun dried for 3–4 days. The two dried samples (Sample 1 and Sample 2) were then put into a mixer grinder, and completely pulverized. The ground sample 1 and sample 2 were then stored in zipper plastic packets. Estimation of crude lipid, crude fiber, ash content (minerals), crude protein content, total free amino acid, and free fatty acid content of the food material of Culicoides and Dasyhelea larvae was done by proximate composition analysis. Proximate composition of the samples were analyzed following the standard methods of the Association of Official Analytical Chemists (AOAC)[21] – crude protein by micro Kjeldahl digestion and distillation, crude lipid by extracting the residue with 40-60°C petroleum ether in a Soxhlet apparatus (Pelican, Chennai, India), crude fiber as loss on ignition of dried lipid-free residue following digestion with 1.25% H₂SO₄ and 1.25% NaOH and ash content by ignition at 550°C (6 hrs) in a Muffle furnace to constant weight. Total free amino acid and fatty acid contents were also measured [22, 23].

Qualitative determination of exoenzyme-producing ability of the gut-associated bacteria in adult midges (Figures 1d-1g)

Battery operated UV light trap (8 W) was used for collecting the biting midges (Site: India, West Bengal, Purba Bardhaman, Burdwan, Keshtopur). The midges were collected in phosphate buffer saline. The collected insects were brought to the laboratory and sorted out quickly to avoid postmortem changes in physiology using a stereozoom microscope Carl Zeiss Stemi 2000 with the help of a fine “000” brush. The species were identified after mounting the genitalia on glass slides [24]. Midges of the genera Culicoides and Dasyhelea were separated and stored in plastic vials filled with phosphate buffer saline. For each species, the sample processing was performed separately. Samples of 0.05 g were weighed in Mettler Toledo Model - JS1203C/A71 (Switzerland) and washed thoroughly with distilled water. As a surface sterilizing agent 70% ethanol was used. The midges were beheaded beneath the stereozoom microscope using a fine needle and taken in an autoclaved plastic container of 2 ml and sterilized distilled volume of 20 times (i.e. 1 ml) of the weighed sample. IKA T10 basic ULTRA-TURRAX Model - T 10B (India) was used to homogenate the sample. The homogenized samples were stored in a refrigerator at 4°C in an autoclaved plastic container. The homogenates were serially diluted (1:10) with sterilized distilled water [25]. A total of three-step dilution 10^-1–10^-3 was prepared for each species. In order to determine the culturable heterotrophic autochthonous aerobic/ facultative anaerobic microbial population, nutrient agar plates were prepared. The plates were stored in a refrigerator (4°C) until further study. A plate for each species having well-separated colonies with distinguished growth is selected to prepare pure culture to get different bacterial isolates. After that, the colony characteristics of each isolate from pure culture were recorded and the colonies were transferred to new culture plates which were stored at 4°C in a refrigerator. Gram staining and endospore staining of bacteria were done. After that measurements of the bacteria were taken underneath compound light microscope (Wild Leitz GMBH, Portugal) using ocular and stage micrometer. For enzymatic analyses bacterial isolates were grown on different culture media and stained: for amylase – cultured in starch agar then stained by Lugol’s Iodine; for protease – cultured in agar with gelatin, peptone, and yeast extract then treatment by HCl + HgCl₂; for cellulase – cultured in bacteriological agar with Carboxymethylcellulose (CMC), then stained by Congo Red; for lipase – cultured in Tributyrin agar and Trybutyrin base. The plates were inspected for any halo zone, if present the zone characteristics were recorded.

RESULTS AND DISCUSSION

Analyses of larval food

The larvae procured from the habitats were identified as Culicoides (Hoffmania) innoxius Sen and Das Gupta, [26] and Dasyhelea (Pseudoculicoides) aprojecta Brahma, Chatterjee and Hazra, [27] upon adult emergence. The result of larval food content (habitat substrata) analysis is depicted in Table 1.

Table 1. Proximate composition of Sample 1 and Sample 2 (g/100g) consumed by the Culicoides innoxius and Dasyhelea aprojectalarvae respectively. Data expressed as mean ± standard error (n = 3).

Qualitative determination of exoenzyme-producing ability of the gut associated bacteria in adult midges (Figure 2)

The species used for enzymatic determination are identified as females of Culicoides (Hoffmania) innoxius Sen and Das Gupta, [26] and females of Dasyhelea (Prokempia) flava Carter, Ingram and Macfie, [28].

In the case of both species colonies of the concentration 10^-1 were overlapped and uncountable. Colonies of concentration 10^-2 were separated with distinct margins and variations, so they were selected for preparation of pure culture (In both Culicoides innoxius and Dasyhelea flava 10^-2 concentration produces eight morphologically distinct colonies) (Table 2). Least numbers of colonies were observed in concentration 10^-3.

Only six isolates of Culicoides innoxius and Dasyhelea flava each (Cu_1-3, Cu_5-7; Da_1-3, Da_5-7) were taken for downstream analysis for better growth in pure culture. Among the six isolates retrieved from both the species (Cu_1-3, Cu_5-7; Da_1-3, Da_5-7) two isolates (Cu₃, Cu₇) were found as Gram-positive while the other four (Cu₁, Cu₂, Cu_5-6) were found to be Gram-negative in the case of Culicoides innoxius, and three isolates (Da_1–3) were found as Gram-positive while other three (Da_5–7) were found as Gram-negative in the case of Dasyhelea flava. All the isolates belonging to Culicoides are bacilli and isolates belonging to Dasyhelea consist of a mixture of cocci form (Da₁) and bacilli (Isolates Da_2–3, Da_5–7). The length and width of bacilli retrieved from Culicoides range from 1.4–2.76 µm and 0.60–0.74 µm respectively (Table 3); the length and width of bacilli retrieved from Dasyhelea range from 1.82–2.8 µm and 0.69–0.74 µm respectively and the diameter of cocci form ranges between 0.62 and 0.64 µm (Table 3). All six isolates from both species were found as non-endospore-forming bacteria.

Determination of enzyme-producing bacteria in adult Culicoides innoxius (Figures 2a–2d; Tables 4 and 5)

Amylase. Halo zones were observed at the periphery of colonies Cu_1-7. So, six isolates were found as amylase positive.

Protease. Halo zones were observed at the periphery of colonies Cu_1-3 and Cu_5-7. So, six isolates were found as protease positive.

Cellulase. Halo zones were observed at the periphery of colonies Cu_1-2. So, two isolates were found cellulase positive.

Lipase. Halo zones were observed at the periphery of colonies Cu_1-3 and Cu_5-7. So, six isolates were found as lipase positive.

Determination of enzyme-producing bacteria in adult Dasyhelea flava (Figures 2e–2h; Tables 4 and 5)

Amylase. Halo zones were observed at the periphery of colonies Da_1-3 and Da_6-7. So, four isolates were found as amylase positive.

Protease. Halo zones were observed at the periphery of colonies Da₁ and Da_6-7. So, three isolates were found as protease positive.

Cellulase. No halo zone was observed around the periphery of any colony. Hence, all six isolates (Da_1–3 and Da_5–7) were found cellulase negative.

Lipase. Halo zones were observed at the periphery of all colonies (Da_1–3 and Da_5–7)  So, six isolates were found as lipase positive.

In past, nutritional and chemical properties of the larval habitats of certain species of Culicoides were evaluated, and categorization of the species of Culicoides was done based on inhabiting high nutrients, moderate to high nutrients, wide nutrient range, moderate to low nutrient, and low nutrient zone [29]. Larvae of Dasyhelea feed on algae and fungi [13]. But carnivorous feeding habit of D. ampullariae Macfie, [30] was also reported [31] which consumes mosquito larvae. There was no record regarding the nutrient contents in the habitat substrata which are consumed by the larvae of Dasyhelea before this study. The larval stages of Culicoides and Dasyhelea actively feed upon the debris, organic matter, and substrata present in their habitat. In this study, proximate composition analysis reveals the number of different nutrients in the food content. High crude fiber quantity has been found in both samples. Moderate quantities of crude protein and crude lipid are also observed which may be crucial for larval maturation. High value of Nitrogen Free Extract (NFE) corresponds to a high amount of carbohydrate which may be important for larval growth. Both types of habitat substrate samples reflect low Free Amino Acids (FAA) and low Free Fatty Acids (FFA) quantity referring to their little requirement for larval development. Finally, a low to moderate amount of minerals (ash content) may be sufficient for sustaining metabolic activity in the larval stages of the Culicoides innoxius and Dasyhelea aprojecta.

Table 2. The colony characteristics of eight bacterial isolates from Culicoides innoxius and Dasyhelea flava.

Table 3. Characteristics of bacteria from pure cultures and their endospore-forming ability.

Table 4. Extracellular enzyme producing ability of different isolates (“+” = positive, “-” = negative).

Table 5. Halo zone characteristics (Diameter: 1-4mm = +, 5-8mm = ++, 9-12mm = +++, 13-16mm = ++++, 17-20mm = +++++ and 21-24mm= ++++++; margin: very diffused = VD, diffused = D, prominent = P, – = negative).