Introduction

Stable flies (Stomoxys spp) have long been considered a major pest of livestock in Kazakhstan and are capable of transmitting pathogens present in infected animals [1]. A high concentration of these flies is observed in almost all regions in Kazakhstan with adult Stomoxys flies emerging in huge numbers following rainfall events in the later months of spring. Farms are a preferred habitat for stable flies where they can be found in large numbers in association with livestock [2, 3]. They interrupt grazing of cattle and cause painful bites, which in turn leads to weight loss in adult and yearling cattle as well as reduced milk yield [4–6]. As a result, farmers have developed strategies to minimize direct contact between animals and biting flies, through pasturing animals in the early morning and in the evening while keeping them in enclosed barns during the daytime when the biting insect numbers are highest.

Stable flies are mechanical vectors for many infectious pathogens [7]. In previous work, we defined the role of Stomoxys species in the transmission of Lumpy Skin Disease Virus (LSDV) [8]. To do so, a constant supply of large numbers of Stomoxys flies reared in laboratory conditions was required. Attempts to produce flies using previously published methods [9, 10] were unsuccessful in obtaining a high yield and as result the aim of this study was to improve the reproduction of three species of Stomoxys using various ambient temperature and relative humidity settings on the different developmental stages of flies.

Materials and methods

Fly catching

Two local farms in the vicinity of the Research Institute for Biological Safety Problems (RIBSP) in Zhambyl oblast, Kazakhstan practising mixed dairy and beef management were examined for the presence of a population of stable fly. Three Stomoxys species were identified, namely S. calcitrans, S. sitiens and S. indica, with S. sitiens considered dominant over the other species.

Farms mentioned above are on the RIBSP balance and no approval was required to access the collection sites.

A fly-catching unit (pyramid) consisting of a cellophane-coated three-piece pyramid-shaped structure was used to collect flies (Fig 1A). The width of the construction at its base was 100 cm and 120 cm height. At the tip of the pyramid, there was a hole of 10 cm in diameter, on which a container was installed to collect flies that enter it as they fly up along the inside of the pyramid. The unit was installed directly onto a dung heap so that the distance between the lower part of the pyramid and the dung was 5–7 cm. Similarly, several units were placed along haystacks. In addition to the unit described above, a commercial fly catching unit “Miniature CDC light trap with UV light” (USA) (Fig 1B) was used. The light trap was suspended from the ceiling of cattle barns. The fly traps were monitored every two hours for the presence of Stomoxys spp. Each batch of newly caught Stomoxys spp was transported immediately to the fly rearing compartment of the Entomology section of the RIBSP for further experimentation. Approximately 780 field collected flies were kept for rearing in glass cages.

Fig 1

a–fly-catching unit (pyramid). b–Miniature CDC light trap with UV light.

Results

Stomoxys sitiens eggs hatched at a lower rate than the other two species (61% vs 68% for Sc and Si; Table 1). No interaction between species and either temperature or humidity was observed indicating the basic shapes of the relationships are similar. Temperature had a significant effect on hatch, however the relationship was curvilinear. Hatch was highest at 32°C and lowest at 27°C. Hatch was higher at 90% humidity (68.3% vs 62.8% at 70% humidity) and temperature and humidity had a significant interaction. Hatch rate was highest for all three species at 32° C and 90% humidity (Table 2).

Table 1

Results of full model GLMM analyses.

Effect	df	% Hatch		% Pupariation		% Adult Emergence
		F	P	F	P	F	P
Species	2,40	4.06	0.0249	0.03	0.9715	2.49	0.0955
Temperature	2,40	25.10	<0.0001	19.25	<0.0001	13.67	<0.0001
Humidity	1,40	6.47	0.0149	0.02	0.8834	1.85	0.1810
Species × Temp	4,40	1.43	0.2423	1.21	0.3210	1.79	0.1500
Species × Humidity	2,40	2.60	0.0869	0.32	0.7277	0.47	0.6256
Temp × Humidity	2,40	21.13	<0.0001	9.15	0.0005	3.50	0.0398

Table 2

Summary of incubation parameters affecting on immature forms of Stomoxys species.

Species	tоС	Stage of development
		Eggs*			Larvae**			Pupae***
		Period, hours/mean	Number of hatched/ mean±SD	% hatched	Period, hours/ mean	Number of pupated/ mean±SD	% pupated	Period, hours/ mean	Number of imago emerged/ mean±SD	% emerged
Relative humidity 90%
SC	32оС	25 (12–30)	119±8.50	79.3	240 (235–250)	49±9.01	33	144 (140–150)	22±6.80	17
SS		28 (13–32)	126±9.45	82	240 (220–255)	32±6.55	21	120 (96–144)	12±5.03	8
SI		22 (15–27)	133±6.02	89	264 (245–276)	51± 12.8	34	120 (100–138)	14±4.72	9
SC	27оС	27 (12–31)	96±13.2	64	216 (204–228)	57±14.0	38	48 (40–72)	32±11.0	21
SS		24 (18–29)	72±11.9	48	216 (200–228)	44±9.60	29	72 (50–84)	30±9.60	20
SI		24 (10–29)	87±9.71	58	240 (216–260)	60±12.2	40	48 (40–56)	29±9.60	19
SC	2оС	30 (18–35)	99±8.50	66	312 (264–327)	59±3.51	39	52 (45–60)	24±7.55	16
SS		26 (19–32)	71±9.01	60,6	288 (238–306)	44±7.02	29	72 (60–85)	30±8.02	20
SI		27 (16–33)	94±9.01	47	288 (230–312)	47±6.50	31	72 (60–84)	27±9.53	18
Relative humidity 70%
SC	32оС	30 (21–33)	102±10.5	68	240 (216–264)	44±10.6	29	96 (90–102)	18±7.55	12
SS		26 (19–29)	89±12.7	59	240 (200–252)	39±4.61	26	96 (86–106)	22±6.55	14
SI		25 (16–30)	93±15.7	62	240 (228–250)	51±9.61	34	96 (90–102)	11±3.21	7.3
SC	27оС	24 (15–29)	84±7.67	56	240 (210–256)	62±11.5	41	72 (65–80)	44±11.1	29
SS		24 (17–26)	93±10.1	62	240 (220–246)	69±14.2	46	96 (84–108)	39±7.51	26
SI		24 (15–28)	75±9.45	50	216 (202–228)	51±9.01	34	72 (66–78)	42±11.3	28
SC	22оС	26 (16–29)	101±9.07	67	288 (240–312)	34±7.02	22	72 (67–96)	9±3.05	6
SS		34 (20–38)	96±5.56	64	288 (246–300)	40±9.53	26	72 (62–96)	13±4.51	8.6
SI		31 (17–37)	112±11.1	75	264 (236–294)	36±10.6	24	72 (65–96)	7±4.04	4,6

Digits in parentheses represents the mean onset and completion time of hatching, pupation and eclosion periods in triplicates.

(*)–A total number of eggs used in the experiment was 150eggs for each Stomoxys species.

(**)—The number of larva pupated from first instar hatched.

(***)The number of imago emerged from viable pupae.

SC—Stomoxys calcitrans.

SS—Stomoxys sitiens.

SI—Stomoxys indica.

No differences were observed in larval survival (hatched -> pupated) among species nor in the interactions between species and temperature or species and humidity. For this reason, species and species interactions were eliminated from the model. In the reduced model, temperature (F = 20.45, df = 2,48, P<0.0001) and the interaction between temperature and humidity (F = 9.59, df = 2,48, P = 0.0003) were both significant. Humidity had no effect on rate of pupariation (F = 0.02, df = 1,48, P = 0.8997). The relationship was curvilinear, the number of larvae successfully pupated was higher at 27°C than either 22°C (t = -4.89 df = 48, P<0.0001) or 32°C (t = 6.17, df = 48, P<0.0001).

For adult emergence, the results were similar to those for larval survival. Neither species nor its interactions with temperature or humidity were significant. In the reduced model, emergence was highest at 27°C (F = 13.01, df = 2,48, P<0.0001). Humidity had no effect on adult emergence (F = 1.34, df = 1,48, P = 0.2524).

Combined method

Combined treatment demonstrated a higher egg to adult survival of Stomoxys spp than the constant condition treatments (46.2% vs 6.7–24.9 for the constant treatments; t = -11.26 - -6.32, df = 56, adjusted P<0.0001). The survival percentage for a total number of 150 eggs incubated under 32°C, 90% RH was above 80% whereas the yield percentage for larvae and pupae maintained under 27°C, 70% RH varied slightly averaging 55% and 48% respectively. The percentage of pupariation in the combined study did not differ from the same treatment in the constant condition study (P = 1.000). Adult emergence was higher than in the comparable treatment in the previous experiment (P ≤ 0.02). The mean quantitative indicators obtained from experiment 3 are shown in Table 3.

Table 3

Quantitative indicators of the development of immature flies using combined incubation parameters in a single life cycle.

Species	Stage of development
	Eggs (32оС, 90% RH)			Larvae (27оС, 70% RH)			Pupae (27оС, 70% RH)
	Period, hours/mean	Number of hatched/mean	% hatched	Period, hours/ mean	Number of pupated/mean	% pupated	Period, hours/ mean	Number of imago emerged/mean	% emerged
SC	29 (15–33)	115±15.04	76.6	240 (204–268)	84±9.53	56	144 (120–156)	62±10.7	41
SS	27 (17–30)	131±9.45	87.3	240 (220–262)	97±11.01	49	132 (108–156)	77±8.73	51
SI	23 (15–28)	120±7.02	80	240 (204–280)	87±8.62	58	120 (108–140)	70±9.01	53

SC—Stomoxys calcitrans.

SS—Stomoxys sitiens.

SI—Stomoxys indica.

Discussion

Blood feeding insects are vectors of infectious diseases of animals [13–16], including lumpy skin disease virus of cattle [8, 17, 18]. In addition, they can cause significant direct damage to livestock and discomfort and stress to companion animals and humans. Such insects include flies of Stomoxys species. To study the role of these flies inhabiting the territory of the Republic of Kazakhstan in the epizootiology of LSD, we have shown that three species of Stomoxys flies are capable of transmitting LSDV from diseased to healthy animals using a fractional feeding method [8]. In connection to this study, the work presented here aimed to study the biology of reproduction of these flies and, based on the results of which, modelling the microclimate for their maintenance and reproduction under laboratory conditions. The created model will subsequently be used in studies evaluating target flies in the role of the mechanical and biological vector of the causative agent of LSD and other vector-borne diseases during spread within the herd and reservation in the natural niche in the interepizootic period.

As the study results show, the developmental biology of all three species of Stomoxys flies was found to be similar. Immature forms of all three species (SC, SS, SI) at the stages of eggs, larva, pupa and imago under the same microclimate conditions developed satisfactorily and similarly. In this study, most of the results obtained align with previous results on the effects of ambient environment on the species’ survival and developmental time under laboratory condition [19, 20]. As for the tested microclimate parameters, we selected two levels of relative humidity, which were maintained at 70% or 90%, and the ambient temperature was selected as equal to 22°C, 27° C, or 32°C. The selection of these parameters was based on studies reported by Kunz, Berry [19], Wang and Gili [21] and Ramsamy [22] Observation of eggs laid and maintained under the indicated microclimate conditions showed that the duration of the hatching period at various temperatures and different humidity were approximately similar to each other, except for a temperature of 22°C, at which this time was extended by 2.6 hours at 90% humidity and by 3.3–6.3 hours at 70% humidity compared with other temperature settings. This observation is in agreement with findings reported by Lysyk [23].

At both humidity indices, the predominantly favorable parameter for the formation of pupae was observed when incubated at 27°C, at which the maximum number of pupae were obtained (35.6% of pupae at 90% humidity and 40.3% of pupae at 70% humidity). The results obtained demonstrate that pupation most favorably occurs at incubation under 27°C and 70% relative humidity. This, in turn, is consistent with previous studies where larvae selected moderate moisture levels in which to pupate avoiding excessively high and low moisture levels [24, 25].

The data obtained during the observation of the final biological development stage of flies demonstrates that humidity and ambient temperature play a decisive role in the active formation and emerging of adult insects. The most suitable indicators of these parameters were humidity equal to 70% and temperature of 27° C.

Analysis of the study results indicates that for the active development of flies and their reproduction in high numbers, a certain microclimate is required [26–28]. At the stage of eggs, an increased humidity of 90% and a relatively high temperature reaching 32°C is favorable and productive for their transition into a larval stage. In this mode, up to 80% of larvae can be obtained from the initial number of eggs laid by an adult fly. Probably, the high humidity level is essential for the formation of larvae. Therefore, they burrow deeper into the medium, where the humidity is higher, from the level at which the eggs were laid.

At the next stage of biological development, when pupation occurs, the demand for higher humidity decreases. A possible explanation for this could be a lower moisture requirement to form a puparium as well as the number of formed pupae becomes relatively small. Therefore, a decrease in humidity at this stage of development stimulates the formation of an increased number of pupae, particularly when incubated at a temperature of 27°C. The benefit of this temperature for the formation of pupae is noted both at high (90%) and low (70%) humidity. Therefore at optimal temperature, humidity is not important (within the range tested). However, at low temperatures, humidity has a significant effect. At high temperature, humidity affected larval survival, but not adult emergence. Effect of temperature on pupariation (22°C, 32°C) did not differ and appeared to be curvilinear. On contrary, pupa formation under 27°C was significantly higher than either P<0, 0001. Temperature and temperature—humidity interaction (marginal) have significant effects on adult emergence P< 0,0001.

Temperature limits above (32°C) or below (22°C) were not evaluated in this study. This was due to an earlier study which showed that constant incubation temperatures at 35°C, 20°C and 15°C resulted in high mortalities in immature Stomoxys forms [20]. For Stomoxys calcitrans, egg to adult survival for all of the constant temperatures was similar to those observed by Florez-Cuadros, Berkebile [29]. Maintaining an incubation microclimate with a humidity of 70% and a temperature of 27° C provided a large population of Stomoxys spp under laboratory settings, reaching 70% or more of the number of stock pupae available.

In addition, the larval medium used in this study has proven suitable for rearing Stomoxys immature forms. Initially, to induce egg laying, a medium was prepared according to Parr (1959) (300g dried cow dung, 150g dried blood granules and 30g sugar). Eggs introduced into this medium demonstrated a meagre percentage of hatching, and those that did hatch did not survive the first instar stage. On reflection, it was hypothesized that dried dung and dried blood became sterile during the medium preparation procedure where ingredients were subjected to complete drying. This is in agreement with findings reported in other studies [30–33] indicating that bacteria present in the natural substrate sustain the viability of stable flies throughout developmental stages. Moreover, production cost is an essential consideration in any stable fly maintenance program. A lack of some costly ingredients in the larval medium used such as a wheat barn, sugar cane bagasse, fishmeal and wood shavings and ammonium bicarbonate may have an effect on the viability of immature Stomoxys flies however this was not evaluated in this study.

Conclusion

To study the biology of the reproduction of Stomoxys flies and the selection of the optimal microclimate for their breeding, seven microclimates were designed and tested, with different temperature conditions and air humidity. Using incubation in double-level microclimate have proven to be efficient at the level of production we have been maintaining.