Fungal biological control agents for integrated management of Culicoides spp. (Diptera: Ceratopogonidae) of livestock

Aim: Entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana had wide host range against insects and hence these are being exploited as fungal bio-pesticide on a large scale. Both fungi are proved pesticides against many crop pests and farmers are well acquainted with their use on the field. Thus, research was aimed to explore the potency of these fungal spores against larval and adult Culicoides midges, a pest of livestock. Materials and Methods: In-vitro testing of both fungal biological control agents was undertaken in Petri dishes against field collected Culicoides larvae, while in plastic beakers against field collected blood-engorged female Culicoides midges. In-vivo testing was undertaken by spraying requisite concentration of fungal spores on the drainage channel against larvae and resting sites of adult Culicoides midges in the cattle shed. Lethal concentration 50 (LC50) values and regression equations were drawn by following probit analysis using SPSS statistical computerized program. Results: The results of this study revealed LC50 values of 2692 mg and 3837 mg (108 cfu/g) for B. bassiana and M. anisopliae, respectively, against Culicoides spp. larvae. Death of Culicoides larvae due to B. bassiana showed greenish coloration in the middle of the body with head and tail showed intense blackish changes, while infection of M. anisopliae resulted in death of Culicoides larvae with greenish and blackish coloration of body along with total destruction, followed by desquamation of intestinal channel. The death of adult Culicoides midges were caused by both the fungi and after death growth of fungus were very well observed on the dead cadavers proving the efficacy of the fungus. Conclusion: Preliminary trials with both funguses (M. anisopliae, B. bassiana) showed encouraging results against larvae and adults of Culicoides spp. Hence, it was ascertained that, these two fungal molecules can form a part of biological control and alternative to chemical control and, therefore, can be inducted in integrated management programs.


Introduction
Using chemical/synthetic pesticides as a single tactic in controlling an important and significant livestock pests have been proved as dangerous as their indiscriminate use have often resulted in problems such as pesticide resistance, pest resurgence, residual toxicity, imbalance in ecological equilibrium, etc. To overcome these problems against any insect pest a strategy involving integrated management in the form of modules is being planned in the present era. Particularly, the integrated pest management (IPM) modules formed against crop pests have yielded tremendous results. However, such IPM modules against livestock pests are yet a dream. Therefore, undertaking the research for development of different biological control agents (BCAs) against important pests of livestock, for inducting in IPM programs, is the need of an hour. Efficacy of two funguses Beauveria bassiana and Metarhizium anisopliae against adult and larvae of houseflies Musca domestica [1][2][3]; against Haematobia irritans [4] and against mosquito larvae [5][6][7] is well-documented. The fungus M. anisopliae (ICIPE-30) and B. bassiana (IMI-391510) spores proved as efficacious in infecting and killing larvae of Anopheles stephensi and Anopheles gambiae under laboratory conditions [8]. It has been demonstrated that infection of adult mosquitoes Culex quinquefasciatus with B. bassiana causes a significant reduction in their survival and disease transmission under field conditions [9].
However, very few studies were undertaken against the Culicoides midges [10,11] and particularly no report is available from India on efficacy of both these funguses against Culicoides midges. Culicoides spp. midges are painful biters, blood suckers and responsible for transmission of many viral diseases of livestock commonly occurring in India. In view of the above-mentioned facts, attempts were made for exploring the potency of these two proven entomopathogenic fungi against Culicoides spp. larvae and adult stages.

Ethical approval
Since prime objective of the study was to develop the fungal candidates as biological control agents for integrated management of Culicoides midges, for which evaluation of two fungal candidates against larvae in drainage channels and against adult midges on the wall of cattle shed were proposed. The institutional animal ethical committee approved the proposed study ensuring that no potential harm toward animal welfare and no direct application of fungus on animal body which will result in any discomfort to the animals.

In-vitro testing of fungal BCAs
Two fungal candidates were evaluated against field collected larvae of Culicoides peregrinus by adapting the techniques described by Busvine [12]. 25 ml test solution prepared from requisite powder of fungal spores, as mentioned in Table-1, was added in each Petri dish. For undertaking in-vitro tests, water used for making dilutions of fugal bio-pesticides were from the same drainage channel, where from the larvae were collected. It was used after harvesting complete larvae and decantation. The idea behind using such water was that, whatever microbial flora and fauna available as food for larvae at natural sites shall be available in petri-dishes too. 25 Culicoides larvae were transferred to each of the test Petri dishes with the aid of pasture pipette. Culicoides larvae were collected from natural breeding sites as per the standard method of larval harvesting [13] by using a simple technique of sedimentation. Mortality was observed after 24, 48, 72, 96 and 120 h of treatment. Experiment was conducted with six replications. Lethal concentration 50 (LC 50 ) values and regression equations were drawn by following probit analysis using SPSS statistical computerized program.

In-vivo trials of BCAs
Trials were undertaken by spraying fungal solution of desired concentration on drainage channels, around the livestock shed, which were positive for larvae of Culicoides spp. Each drainage channel was labeled with nameplate for a particular fungus and concentration. Each drainage channel used for spraying fungus solution was selected for 10 m length and then sealed at both proximal and distal ends. The purpose of sealing was to cease the new addition of larvae accruing from incoming water from the proximal end of the channel. Larval count was recorded before and after spraying at 24 h interval till larval count reaches negligible or zero level. Larval count was recorded by using the standard method of larval harvesting [13] in a weighed quantity of substrate collected from the drainage channel.

In-vitro and in-vivo testing against adult C. peregrinus midges
Trials were conducted as: • Trial A (In-vitro): Trials of each fungus were conducted in plastic beakers of 1000 ml volume (10 cm diameter and 15 cm height). Filter paper treated with dried fungal powder @ 750 mg/900 cm (30 cm 2 × 30 cm 2 ) area was adhered to sides and bottom of the beaker, in which 25 blood engorged females were released, and it was closed with Both fungus in the solution form prepared by mixing 5 g powder+ 5 ml milk + 1000 ml of water, applied on marked square of 2 × 2 feet (60 cm × 60 cm) @ 30 ml/m 2 area. Culicoides midges resting on such sites were collected with the help of insect aspirator and were maintained in the laboratory for observing the mortality. Dead cadavers of midges from treated and control squares were incubated for fungal growth. Each observation was replicated 6 times.

Processing of dead midges
To wipe out the fungal spores adhered to the body of dead cadavers of Culicoides midges collected from trials A and B, were washed with several rinsing of normal water. Such cadavers were rolled in the moist tissue paper and kept in desiccators having humidity level of 75% at room temperature. The humidity levels were created by adding NaCl solution (40 g/100 ml of water) at the bottom of desiccators. Dead cadavers of Culicoides were observed for growth of fungus under zoom stereoscope daily for more than 15 days.

Effect of fungus on larvae and adult Culicoides midges in vitro and in vivo
The LC 50 values estimated against Culicoides larvae were 2692 mg and 3837 mg (10 8 cfu/g) for B. bassiana and M. anisopliae, respectively by drawing the probit lines (Tables-2 and 3; Figures-1 and 2). Data from Table-1 indicated that fungal infection to Culicoides larvae resulted in mortality and therefore larval count was consistently showing decreasing trend, although, daily there was the addition of newer larvae hatched from eggs laid by Culicoides midges. However, 7 days post infection, the larval count was resuming to its original number as observed on day 0 and both funguses found effective for maximum 7 days, necessitating its innudative application. Under trial "A" death of adult Culicoides midges was caused by both the fungi within 24-48 h, while death of midges in the control was observed after 96 h and after death growth of fungus was very well observed on the dead cadavers of infected midges (Figures-7-12). Close look to Figures-7 and 10         The purpose of the present study was not to compare the efficacy of two fungi studied and data was not generated to that context, but grossly it can be opined that, under prevailing environmental conditions of      Marathwada (Parbhani) region of Maharashtra state, M. anisopliae fungus found comparatively better choice in terms of larval mortality and growth of fungus in the body of adult midges. Place of present study, Marathwada region lies between Latitude 17°35'N-20°40'N Longitudes 70°40'-78°15' E, MSL -40.9 m, in the Deccan Plateau Zone, is basically an intense agrarian region. Climatologically it was categorized as semi-arid on an annual basis. It is humid to per-humid during monsoon, sub-humid to semi-arid during winter and arid during the summer season. The corresponding distribution pattern of annual rainfall (500-1100 mm) is 75-85, 10-15 and 6-10%, respectively. The wet monsoon (weeks 23-44, 4 th June-4 th November) period alternates with long rain free cold winter (weeks 45-9, 5 th November-4 th March). Minimum temperature ranges between 12°C and 18°C and maximum between 40°C and 45°C is observed during the summer (weeks 10-22, 5 th March-3 rd June), having appreciable temporal and spatial variability, a typical characteristics of semi-arid climate [14].
Entomopathogenic fungi are widely distributed throughout the fungal kingdom, although the majority of them occur in the Deuteromycotina and Zygomycotina group of which M. anisopliae and B. bassiana has wide host range and hence these are being exploited as fungal bio-pesticide on large scale. The mode of action on generalized line of fungus against insects was narrated by Goettel and Inglis [15]. According to their study, most common route of host invasion is through the external integument (contact with conidiospores, i.e., conidia). Though, infection via digestive tract is possible. Conidia attach to the cuticle, germinate, and penetrate the cuticle with the help of enzymatic degradation and pressure of the germ tube [16,17]. Once in the hemocoel, the mycelium grows throughout the host, forming hyphal bodies called blastospores. Death of the insect is often due to a combination of action of fungal toxins, physical obstruction of blood circulation, nutrient depletion and/or invasion of organs. After the host dies, hyphae usually emerge from the cadaver and under suitable abiotical condition, conidia are produced on the exterior of the host. These are then dispersed by wind or water. The infection may also take place via the respiratory system [18]. After infection, free-floating, yeast-like hyphal bodies are produced and spread throughout the hemocoel [19]. Similarly, data from Table-3 also indicates that LC 50 values of both fungi and their corresponding LD 50 values are indicative of their safety and therefore can be recommended for induction in IPM programs.
It will be not be out of context to review mode of action and efficacy of both fungi judged by various authors against the mosquito larvae as detailed by Scholte et al. [20] in reference to present study.

B. bassiana
Conidial dry powder when poured on water surface of breeding sites of mosquito , conidia being hydrophobic, thus floating on the water surface, and contact mosquito larvae that feed below the surface mainly at the tip of the siphon, blocks the respiration and thereby kills the larvae. In adults conidia enters through the spiracles, get germinated, invades the walls of the tracheae, and subsequently were thought to release a toxin that kills the adults [21]. Subsequent studies about the toxins of this genus confirmed were beauvericin, bassianin, bassianolide, beauverolides, and tenellin from B. bassiana, [22]. Hart and MacLeod [23], who recorded the germination of Beauveria conidia described the requirement as RH above 94%, however infection does not appear to be dependent on temperature [16]. In laboratory tests against adult Culex tarsalis, Culex pipiens, Aedes aegypti, Ochlerotatus sierrensis, Ochlerotatus nigromaculis (Ludlow), and Anopheles albimanus, conidia of B. bassiana produced 100% mortality within 5 days after exposure [21]. Sections of infected adults, fixed and imbedded in paraffin immediately after death, revealed some mycelium in regions surrounding the main tracheal trunks. From these observations, [21] inferred that the conidia appeared to be entered through the spiracles, germinated, invaded the walls of the tracheae, and subsequently were thought to release a toxin that killed the adults.
In the light of these observations, it can be stated that fungus infection studied against Culicoides midges appears to be resulted in producing similar type of pathogenesis. When fungus was applied at the resting sites of Culicoides, they picked up the infection, may be through tarsi, and death took place within 24 h with ample growth of fungus on the body after 10 days period ( Figures-9 and 12). Thus, spray of B. bassiana fungus on resting sites of Culicoides, can be the effective way of biological control of adult Culicoides midges. Beauveria is one of the most frequently isolated entomogenous fungal genera and has a cosmopolitan distribution, though the genus has a very broad host range [24]. In three small-scale outdoor tests with conidia of B. bassiana, reductions of 82, 95 and 69% were found on C. pipiens larvae and pupae after 2 weeks [21]. Similar type of results was obtained in regards to reduction in Culicoides larvae when B. bassiana fungus was applied to drainage channels in the present field study (Table-1). Despite consistent results obtained in the lab, on-field results were vague and inconsistent. Two drawbacks of B. bassiana fungus mentioned in the literature are, (1) Susceptible species were prone to infection only shortly after moulting. If the infection occurred shortly before moulting, the mycelium was lost within the moult, (2) a problem associated with using conidia is that they have no residual effect. They germinate in mosquito habitats even when not in contact with larvae. In the present project field trials, too, it appeared that fungus needs innudative application without showing much residual effect (maximum up to 7 days).

M. anisopliae
According to Boucias and Pendland [25], fungus has a large host-range, including arachnids and five orders of insects. On terrestrial insects, the life cycle begins with conidia attaching to the host cuticle, forming an appressorium, followed by a penetration peg to enter the cuticle. After entering the hemocoel, hyphae formed that produces and releases toxins, killing the host within 4-16 days. These toxins released are destruxins, swainsinone, and cytochalasin C [22]. If the conditions are warm and moist, conidiophores will grow through the cuticle to cover the insect with conidia. At breeding sites, if conidia are applied on the water surface, larvae contact to conidia through perispiracular valves during their air intake. The fungus germinates and penetrates into the respiration siphon, blocking the breathing mechanism [26,27]. Plugging of the spiracles usually leads to death before significant invasion of the hemocoel occurs, so hyphal body formation is minimal. The optimal growth temperature for most strains is 27-28°C and RH 92% [16]. According to the literature, M. anisopliae has several characteristics that make it interesting as a microbial mosquito control agent. It has advantage that this do not germinate in the mosquito environment until actual exposure to a host and its resulting persistence in the environment, as well as the fact that its effect is not limited to periods of host moulting, which has taken this fungus up as compared to B. bassiana, and hence make this fungus a very promising control agent [24]. One drawback mentioned in the literature in regards to use of M. anisopliae for mosquito control is that conidia are not produced on submerged fungus killed larvae, and hence inundated releases are required. Perhaps this could be reason that conidia may not be getting produced on dead Culicoides larvae and hence in the present study too, innudative releases of fungus on drainage channels was felt essential after 7 days PI (Table-1).

Conclusion
Efficacy of both the fungus B. bassiana and M. anisopliae were proved against adult and larvae of housefly, M. domestica (Diptera: Muscidae) [1,2] and against H. irritans [4], against mosquito larvae [5][6][7]. All these observations indicate that both fungus candidates are proved as efficacious against many livestock pests, with the addition of the present study indicating their efficacy against Culicoides adults/larvae. Thus, use of these two fungus candidates is the novel weapon in the hands of farmers for covering management of range of insect pests.

Author's Contributors
BWN being PI of the research project conceptualized and implemented the technical program. BWN collected, processed and analyzed the data and submitted it in the form of present research article. PRS being Co-PI was involved in the finalization of technical program and involved in validation and confirmation of data and taxonomy related work of adult and larval Culicoides midges. PCH, Junior research fellow in the project and specialized in Microbiology, carried all laboratory work related to fungus and also contributed for undertaking In-vivo and In-vitro trials. All authors read and approved the final manuscript.