Absence of abdominal pigmentation in species of Culicoides Latreille Subgenus Trithecoides Wirth &amp; Hubert: An obstruction to age-grading

doi:10.21203/rs.3.rs-3564074/v1

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Absence of abdominal pigmentation in species of Culicoides Latreille Subgenus Trithecoides Wirth & Hubert: An obstruction to age-grading

https://doi.org/10.21203/rs.3.rs-3564074/v1

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Examination of gravid female specimens of nine species of Culicoides subg Trithecoides, viz C. acanthostomus, C. anophelis, C. flavescens, C. paraflavescens, C. flaviscutatus, C. inornatithorax/flaviscutatus, C. palpifer, C. palpifer/neopalpifer, and an undescribed species, revealed that none possessed the dark abdominal pigmentation characteristic of gravid and parous females of species belonging to other subgenera of Culicoides. The lack of abdominal pigmentation was supported by data from 68 separate collections from 7 countries in southern Asia and Australasia containing more than 4100 female specimens belonging to C. subg Trithecoides, of which none had pigment. This lack of pigmentation is likely widespread within this subgenus and may also be lacking in other subgenera. The lack of abdominal pigmentation hinders the visual age grading of specimens belonging to this subgenus, which will have implications for survival studies and the selection of specimens for viral isolation and detection.

Trithecoides

parous rate

gravid

parity

Culicoides anophelis

Culicoides flavescens

Culicoides acanthostomus

Culicoides paraflavescens

Culicoides flaviscutatus

Culicoides palpifer

Culicoides spp. Latreille (Diptera: Ceratopogonidae) are notorious hematophagous midges responsible for transmitting a wide range of arboviruses, protozoans, and nematodes (Meiswinkel et al. 2004). In common with other biological vectors of animal pathogens, survival of the insect vector is crucial to the epidemiology of the disease (Work et al. 1991). One of the most widely used methods of estimating the survival rate of haematophagous flies is the calculation of the parous rate, which estimates the proportion of adult females which survive over the period between successive feedings on a host, known as the gonotrophic cycle (Macdonald 1952). Calculating the parous rate requires accurate counts of the number of nulliparous and parous females in the population, which in turn requires a reliable means of recognizing females in these physiological states. Recognition of parous females is also useful when collecting evidence of the vector status of a species as detection of the pathogen in field-collected insects is one requirement for proving its vector status (Anon 1967), and it is the parous females that have previously fed on blood that are the most likely to be harbouring such pathogens. Of the range of available methods of distinguishing parous and nulliparous female haematophagous flies, the presence of a burgundy red pigment in the abdomen of parous and gravid females of some species of Culicoides (Dyce 1969) has dominated studies of vector biology of species belonging to this genus (Nelson and Scrivani 1972; Phelps et al. 1982; Walker 1977; Birley and Boorman 1982; Mullens and Schmidtmann 1982; Gubbins et al. 2008; Hartemink et al. 2009; Braverman and Mumcuoglu 2009; Guis et al. 2012)

Despite the overwhelming adoption of Dyce’s method of recognizing parous females in studies involving a wide range of species of Culicoides (Goeffredo et al. 2021), very few species have actually undergone studies like those undertaken by Dyce (1969) to confirm the association between parity and pigmentation (Akey and Potter 1979; Linley and Braverman 1984, 1986; Braverman and Mumcuoglu 2009). Conversely, some studies have reported low numbers of pigmented females of Culicoides imicola Kieffer (Braverman and Mumcuoglu 2009) and other species belonging to C. subg. Avaritia Fox (Harrup et al. 2013) in emergence traps suggests that newly emerged i.e., nulliparous females, of these species may also possess pigment. Conversely, Goefreddo et al. (2021) recently reported that a small proportion of females of several African and European species did not develop pigment following ingestion of a small blood meal. Other reports indicate that the distinction between pigmented and unpigmented specimens of Culicoides subimmaculatus Lee and Reye was difficult to assess, even in gravid females whose parity is beyond question (Kay and Lennon 1982). The present work reports on the absence of pigmentation in specimens of several species of C. subg. Trithecoides Wirth & Hubert.

Culicoides midges were collected using CDC-style light traps modified to collect specimens directly into the liquid. Specimens were collected directly into either 70% ethanol or water, to which a few drops of soap were added, then transferred into 70% ethanol the following morning. Traps were typically run from dusk to dawn and set opportunistically at various sites across Papua New Guinea, Thailand, China, Indonesia, Timor-Leste, Solomon Island, and India (Table 1).

To observe the development of eggs in individual specimens, blood-engorged females belonging to C. subg. Trithecoides were orally aspirated from cattle and kept alive following the techniques of Harsha and Mazumdar (2015) until the eggs had developed fully.

Culicoides spp. were examined under a stereo microscope and identified as belonging to C. subg. Trithecoides by the relative size of the second radial cell, wing patterning, and colouration of the thorax and legs. Specimens from some collections were able to be identified as species by comparison with descriptions in Wirth and Hubert (1989) and with reference specimens. All females were graded and counted based on abdominal pigmentation and contents. Gravid females were dissected to count the number of eggs, and then these and a series of representative specimens of other females in the traps were mounted onto slides following techniques of Wirth and Marston (1968) or Bellis et al. (2013) and identified to species using the keys and descriptions of Wirth and Hubert (1959, 1989), Yu et al. (2006) or Nandi and Mazumdar (2015).

Slide-mounted specimens of species belonging to C. subg. Trithecoides that are lodged in the National Arbovirus Monitoring Program Insect Collection, Darwin, Northern Territory, Australia, were also examined, and any gravid females were examined for the presence of abdominal pigmentation.

More than 5300 specimens belong to C. subg. Trithecoides were examined from 68 collections from 7 countries (Table 1). Of these, 4158 females had no abdominal contents, and 21 were gravid. No specimens, including all gravid females, exhibited any trace of dark pigment in their abdomen, so they could not be sorted into nulliparous or parous age grades (Fig. 1). The species and number of unfed, fed and gravid females represented in these collections are presented in Table 1. Similarly, the 24 live, blood-fed females belonging to Culicoides anophelis Edwards did not exhibit any traces of abdominal pigmentation even 4 to 5 days after oviposition, although egg development and egg laying was observed in all of these specimens.

While some species within this subgenus can be reliably identified using thorax and leg colouration, many others are difficult to identify to species without examining characters that are only visible on slide-mounted specimens and as only a small proportion of specimens were mounted, many specimens remain unidentified to species. Specimens that were mounted and accurately identified were referable to Culicoides barnetti Wirth and Hubert, Culicoides flavescens Macfie, Culicoides flaviscutatus Wirth and Hubert, Culicoides anophelis, Culicoides parahumeralis Wirth and Hubert, Culicoides palpifer Sen and Das Gupta, Culicoides paraflavescens Wirth and Hubert, Culicoides gewertzi Causey, Culicoides culiciphagus Wirth and Hubert, Culicoides acanthostomus Wirth and Hubert and C. Trithecoides sp no 1 sensu Dyce et al. 2007 (Table 1). Three specimens from northern India ran to C. flaviscutatus in the key of Wirth and Hubert (1989) and to C. inornatithorax Das Gupta in the key of Nandi and Mazumdar (2015), but as the former species has not previously been reported from India and we have no reference specimens of C. inornatithorax we were unable to confirm the identity of these specimens. Similarly, we could not definitively identify a specimen from Hainan Province, China, which did not run cleanly to any species in the keys of Yu et al (2006) but matched descriptions of both C. palpifer and C. neopalpifer.

Egg counts of gravid female C. acanthostomus, C. anophelis, C. flavescens, C. paraflavescens, C. flaviscutatus, C. inornatipennis/ flaviscutatus, C. palpifer, C. palpifer/neopalpifer and C. Trithecoides sp no 1 sensu Dyce et al. (2007) are presented in Table 2. It was difficult to obtain accurate counts of the number of eggs in slide mounted specimens, particularly where large numbers of eggs were present, and counts probably underestimate the true number Nevertheless, it appears that the fecundity of C. anophelis was relatively higher than the other species examined. None of these gravid females examined exhibited any trace of pigment.

Species of C. subg. Trithecoides have so far not been implicated in the transmission of any pathogen. The potential for involvement of the mosquito-biting species of the Anophelis Group in the transmission of mosquito-borne diseases (Sinton and Little 1925; Laird 1946) remains unclear as there are conflicting reports regarding the source of the blood meal. Sen and Das Gupta (1959) and Das Gupta (1964) believed that this midge feeds on mosquito hemolymph rather than its vertebrate blood meal but recent video evidence from Ma et al. (2013) of C. anophelis feeding on an engorged mosquito clearly shows the midge with a bright red abdomen indicative of ingestion of vertebrate blood. The lack of evidence incriminating species of C. subg. Trithecoides as vectors has negated the demand for a means to recognize parous females for studies on parous rate or screening for pathogens and there are consequently no published reports for any species of the subgenus, even though several species have been reported as feeding on cattle (see Wirth and Hubert 1989).

Despite relatively recent regional taxonomic revisions incorporating the species of C. subg. Trithecoides from SE Asia (Wirth and Hubert 1989), China (Yu et al. 2006) and India (Nandi and Mazumdar 2015), difficulties were experienced in identifying specimens from northern India and southern China. This suggests that some taxonomic revision of the subgenus in Asia is warranted, preferably one that includes all species present in Asia and employs integrative taxonomy.

The absence of pigment in gravid specimens of C. flavescens, C. acanthostomus, C. anophelis, C. paraflavescens, C. inornatipennis/ flaviscutatus, C. palpifer, and C. Trithecoides sp no 1 indicates that this pigment does not develop in at least some specimens of these species. Linley and Braverman (1984) proposed that large blood meals or egg masses reduced circulation of hemolymph to extremities of the abdomen which resulted in an accumulation of ommochrome waste products producing the pigment observed in parous specimens. More recently, Goeffredo et al. (2021) suggested that pigment production was more likely to be related to egg formation than blood feeding as pigmentation has been reported in autogenous individuals that had not taken a blood meal (Birley and Boorman, 1982; Tyndale-Biscoe, 1984). Goeffredo et al. (2021) additionally observed that a low number of individuals of several species did not produce pigment following ingestion of a relatively small blood meal, which did not have a large batch of eggs, supporting Linley and Braverman’s (1984) earlier claim that the size of the blood meal and subsequent egg mass affected pigment production. However, the large number of eggs that filled the abdomen in several of our specimens indicates the lack of pigmentation in species of C. subg. Trithecoides are not due to small egg mass, as Goffered et al. (2021) suggested. The total absence of pigment in 68 separate collections containing more than 4100 female specimens of several species provides supporting solid evidence that this phenomenon does not occur in the more common species of C. subg. Trithecoides in southern Asia and PNG may be more widespread in the genus.

The lack of pigmentation in some species belonging to C. subg Trithecoides stands as an exception to Dyce’s method of determining parity so that studies of these species will require an alternative method of age-grading female specimens.

Acknowledgement

We acknowledge the Head, Department of Zoology, The University of Burdwan for providing laboratory facilities.

Authors’ contributions

SMM helped design the study, insect sampling, and data analysis. RH, AM, JL, RHu, AT, AHW, FI contributed to specimen collection. AM ensured the entire process was correct and contributed to the final write-up. GB designed the study, identified most of the specimens, analysed the data and wrote the initial draft of the manuscript.

Data availability

All data generated during the study were available in this manuscript.

Conflict of interest

There has been no conflict of interest amongst the authors. The manuscript has been submitted unanimously by all the co-authors.

Ethics approval

In this study no animals/ humans were subjected to any observational or experimental trial.

Funding

No funding was received for this study.

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Table 1 Collections of Culicoides subg. Trithecoides examined for presence of abdominal pigmentation

Site	Tentative identification (unmounted)	empty, no pigment	fed	gravid	male	total	representatives identified
Papua New Guinea, Western Province, Gre via Kiunga, 06.01238S 141.18238E; 2-6.Jun.2011, Lt Tp., S.Fruean & N.Harris	Trithecoides sp no 1 sensu Dyce et al (2007)	17	1	0	0	18	-
Papua New Guinea, Western Province, Gre via Kiunga, 06.01238S 141.18238E; 2-6.Jun.2011, Lt Tp., S.Fruean & N.Harris	Trithecoides sp no 1 sensu Dyce et al (2007)	45	5	0	1	51	-
Papua New Guinea, Milne Bay Prov, Baba via Alotau, 10º18.882'S 150º22.939'E 27.Nov.2009, LT, L. Halling	Trithecoides sp	22	1	0	0	24	1 palpifer, 4 Trithecoides sp no 1, 8 barnetti
Papua New Guinea, Western Prov. Indorodoro Site 2 8º03.267’S 141º41.038’E 27.May.2009 B.Cookson & N.Harris	Trithecoides sp	90	0	0	32	122	5 Trithecoides sp no 1
Papua New Guinea, Sandaun Prov., Krisa 2°51.034’S 141°17.585’E 30.Nov-2.Dec.2009 LT N. Harris	Trithecoides sp	10	0	0	2	12	4 palpifer, 12 Trithecoides sp no 1
Papua New Guinea, Sandaun Prov, Ossima Mission via Krisa, 2.91782S, 141.2976E, 24-27.Apr.2016, D.Tenakanai	Trithecoides sp no 1 sensu Dyce et al (2007)	4	1	0	1	6	-
Papua New Guinea, East Sepik Prov. Wirui Mission, via Wewak 3°34’692’S 143°38.767’E 22.Oct.2007 LT A. Rice	Trithecoides sp no 1 sensu Dyce et al (2007)	6	0	0	0	6	-
Papua New Guinea, Madang Province, Stewart farm, S 4.96325; E 145.77666, 21-24.Apr.15, N.Harris	Trithecoides sp no 1 sensu Dyce et al (2007)	419	2	0	58	481	-
Papua New Guinea, Madang Province, Stewart farm, S 4.96325; E 145.77666, 21-24.Apr.15, N.Harris	Trithecoides sp no 1 sensu Dyce et al (2007)	0	0	0	1	1	-
Thailand, Trang cowshed, 7.83065°N, 99.33763°E, LT, 20:00PM, 9.Jun.2012; No3	C. barnetti	110	10	0	3	123
Thailand, Trang cowshed, 7.83065°N, 99.33763°E, LT, 20:00PM, 9.Jun.2012; No3	C. gewertzi	39	11	0	0	50	1 C. gewertzi
Thailand, Trang cowshed, 7.83065°N, 99.33763°E, LT, 20:00PM, 9.Jun.2012; No3	C. gewertzi	4	1	0	0	5	1 C. gewertzi
Thailand, Trang cowshed, 7.83065°N, 99.33763°E, LT, 20:00PM, 9.Jun.2012; No3	C. barnetti	9	3	0	0	12
Thailand, Trang cowshed, 7.83065°N, 99.33763°E, LT, 20:00PM, 9.Jun.2012; No3	Trithecoides sp.	6	3	0	0	9	1 C. flavescens
China, Hainan, Danzhou, Nada, net near penned buffalo 1800-1830hr, 15.Dec.2010, D. Cai	Trithecoides sp.	78	0	0	0	78
China, Hainan, Danzhou, Nada, net near penned buffalo 1800-1830hr, 15.Dec.2010, D. Cai	Trithecoides sp.	18	0	0	0	18
China, Hainan, Lao Cheng site 1, via Haikou, 19.96°N, 110.11°E, LT, 15.OCT.2015, J-H Li & D-C Cai	C. anophelis	636	0	0	0	636	2 C. anophelis
China, Hainan, Lao Cheng site 1, via Haikou, 19.96°N, 110.11°E, LT, 15.OCT.2015, J-H Li & D-C Cai	C. palpifer	79	0	0	0	79
Indonesia, West Java, Cibinong, Sanja (LIPI), 6.51°S 106.87°E, 1.June.2011, net near 40 penned cattle 1730-1830 hr, G.Bellis	Trithecoides sp.	52	2	0	1	57
Indonesia, West Java, Mekar Wangi, Cikarang, Bekasi, 6.25667°S 107.15611°E, 28.May.2011, net near 12 penned cattle 1730-1745 hr, G.Bellis	Trithecoides sp.	8	5	0	0	18
Indonesia, West Java,Mekar Wangi, Cikarang, Bekasi, 6.25667°S 107.15611°E, 30.May.2011, light trap near 12 penned cattle, G.Bellis	Trithecoides sp.	16	4	0	0	24
Indonesia, Java, Yogyakarta, Kediri, Ngadiluwih, Rembang Kepuh, 7.89°S 112.01°E,, LT over Cattle pen, 18.Jul.2011, A.Wardhana	Trithecoides sp.	151	23	0	0	174
Indonesia, Java, Yogyakarta, Kediri, Ngadiluwih, Rembang Kepuh, 7.89°S 112.01°E,, LT over Cattle pen, 19.Jul.2011, A.Wardhana	Trithecoides sp.	132	3	0	1	139
Indonesia, Java, Yogyakarta, Kediri, Ngadiluwih, Rembang Kepuh, 7.89°S 112.01°E,, LT over Cattle pen, 25.Jul.2011, A.Wardhana	Trithecoides sp.	5	0	0	0	5
Indonesia, Java, Yogyakarta, Desa Ringinsari, Kecamatan Kandat, Kabupaten Kediri, LT over Cattle pen, 24.Jul.2011, A.Wardhana	Trithecoides sp.	7	0	0	0	7
Indonesia, Java, Yogyakarta, Desa Ringinsari, Kecamatan Kandat, Kabupaten Kediri, LT over Cattle pen, 23.Jul.2011, A.Wardhana	Trithecoides sp.	128	0	0	1	129
Indonesia, Java, Yogyakarta, Kampung Ternak, Desa Ringinsari, Kecamatan Kandat, Kabupaten Kediri, LT over Cattle pen, 24.Jul.2011, A.Wardhana	Trithecoides sp.	12	0	0	0	12
Indonesia, Java, Yogyakarta, Desa Ringinsari, Kecamatan Kandat, Kabupaten Kediri, LT over Cattle pen, 27.Jul.2011, A.Wardhana	Trithecoides sp.	28	2	0	8	40
Indonesia, Java, Yogyakarta, Dusun Rembang Kepuh, Desa Rembang Kepuh, Kecamatan Ngadiluwih, Kabupaten Kediri, LT over Cattle pen, 19.Jul.2011, A.Wardhana	Trithecoides sp.	12	3	0	0	18
Indonesia, Java, Yogyakarta, Kediri, Kepung, Kampung Baru, 7.17°S 112.72°E, LT over Cattle pen, 21.Jul.2011, A.Wardhana	Trithecoides sp.	5	1	0	0	7
Indonesia, Java, Yogyakarta, Kediri, Kepung, Kampung Baru, 7.17°S 112.72°E, LT over Cattle pen, 20.Jul.2011, A.Wardhana	Trithecoides sp.	12	0	0	1	13
Indonesia, Java, Yogyakarta, Kediri, Kepung, Kampung Baru, 7.17°S 112.72°E, LT over Goat pen, 21.Jul.2011, A.Wardhana	Trithecoides sp.	2	0	0	0	2
Indonesia, Java, Yogyakarta, Desa Ploso Kidul, Kecamatan Plosoklaten, Kabupaten Kediri, LT over Cattle pen, 14.Mar.2012, A.Wardhana	Trithecoides sp.	3	2	0	0	7
Indonesia, Java, Yogyakarta, Desa Ploso Kidul, Kecamatan Plosoklaten, Kabupaten Kediri, LT over Cattle pen, 14.Mar.2012, A.Wardhana	Trithecoides sp.	1	0	0	0	1
Indonesia, Java, Yogyakarta, Desa Ploso Kidul, Kecamatan Plosoklaten, Kabupaten Kediri, LT over Cattle pen, 14.Mar.2012, A.Wardhana	Trithecoides sp.	2	0	0	3	5
Indonesia, Java, Yogyakarta, Desa Ploso Kidul, Kecamatan Plosoklaten, Kabupaten Kediri, LT over Cattle pen, 15.Mar.2012, A.Wardhana	Trithecoides sp.	6	0	0	0	6
Timor-Leste, Lautem, Fuiloro cattle yards, 21.Jun.2004, E Watkins green	Trithecoides sp.	26	1	0	0	28
Timor-Leste, Bobonaro, Maliana, Suco Holsen, Ald. Solugalu, 8º59.463'S; 125º11.888'E, 19.Jun.2002, E. Watkins,	Trithecoides sp.	7	0	0	1	8
Timor-Leste, Bobonaro, Maliana, Tunubibi, E125.08.413; S08.58.636, 17.May.2010 B. Cookson	C. parahumeralis	1	0	0	0	1
Timor-Leste, Bobonaro, Maliana, Tunubibi, E125.08.413; S08.58.636, 17.May.2010 B. Cookson	Trithecoides sp.	12	8	0	0	20
Timor-Leste, Oecusse, Pante Macassar 4.Dec.2002 A.Moss, LT over 3 tethered cows	Trithecoides sp.	15	4	0	1	20
Timor-Leste, Cova Lima, Suai, 5.May.2002 G.Bellis	Trithecoides sp.	212	44	0	4	260
Timor-Leste, CovaLima, Suai, 16-18 Jun 2004 E. Watkins	Trithecoides sp.	8	0	0	0	8
Timor-Leste, Suai, CCT compound, 13-14.May.2010 B. Cookson	Trithecoides sp no 1 sensu Dyce et al (2007)	16	6	0	0	22
Solomon Is, Malaita, Kwailatutu, 8°56’24’’S; 160°46’43”E, Green LED trap in cattle paddock, 18.May.2010, L. Halling LH712	Trithecoides sp no 1 sensu Dyce et al (2007)	56	4	6	2	72	Trithecoides sp no 1
Solomon Islands, Choiseul Province, Choiseul Bay Ag Stn, -6.6959°S, 156.4195°E, light trap near 4 penned pigs, m, 16.Feb.2012, G.Bellis	Trithecoides sp no 1 sensu Dyce et al (2007)	107	83	0	0	190
Solomon Islands, Western Province, Gizo, 6 mile cattle farm, -8.0652°S, 156.7823°E, light trap over penned pig, m, 21.Feb.2012, G.Bellis	Trithecoides sp no 1 sensu Dyce et al (2007)	61	100	0	0	261
Solomon Islands, Guadalcanal, Tenavatu, -6.7129°S, 156.4094°E, light trap in riparian jungle next to cattle paddock, m, 27.Feb.2012, G.Bellis	Trithecoides sp	126	5	0	3	134	1 C. culiciphagus, 1 C. barnetti
Thailand, F pig	C. barnetti/C. manukunari	4	10	0	0	23
Thailand, F pig	C. elbeli	0	1	0	0	1
Thailand, F pig	C. flaviscutatus	4	3	0	0	10
Thailand, F pig	C. palpifer	17	11	0	0	39
Thailand, F pig	Trithecoides	1	1	0	0	3
Thailand, F pig	C. flavescens	4	0	1	0	5
China, Hainan, Qiong Hai, Hui Shan Town, Jia LueVillage, LT near 2 pigs, 2.Oct.16, Wei-zhu Jiang	C. flaviscutatus	1	4	0	0	5
China, Hainan, Qiong Hai, Hui Shan Town, Jia LueVillage, LT near 2 pigs, 2.Oct.16, Weizhu Jiang	C. palpifer	4	2	0	0	6
China, Hainan, Haikou, Yun Long Town, Mai Hong Village, LT in chicken and duck farm, 8.Dec.2016, J-H. Li	C. flaviscutatus	27	0	0	0	27
China, Hainan, Haikou, Yun Long Town, Mai Hong Village, LT in sheep farm, 8.Dec.2016, J-H. Li	C. anophelis	46	0	0	0	46
China, Hainan, Haikou, Yun Long Town, Mai Hong Village, LT in sheep farm, 8.Dec.2016, J-H. Li	C. flaviscutatus	219	3	0	0	222
China, Hainan, Ding An, Nanlihu Wetland Park, LT 9.Sep.2016, J-H. Li	C. flaviscutatus/ C. parabarnetti	1	0	0	0	1
China, Hainan, Wuzhishan Nature Reserve, LT near creek in forest, 29,Dec.2016, J-H. Li	C. flavescens	1	0	0	0	1
China, Hainan, Wuzhishan Nature Reserve, LT near creek in forest, 29,Dec.2016, J-H. Li	C. palpifer	1	0	0	0	1
China, Hainan, WuzhishanNature Reserve, LT near gate and penned chickens, 29,Dec.2016, J-H. Li	C. huberti	1	0	0	0	1
China, Hainan, WuzhishanNature Reserve, LT near gate and penned chickens, 29,Dec.2016, J-H. Li	C. baisasi/ C. flavescens	0	0	1	0	1
China, Hainan, Wuzhishan Nature Reserve, LT near gate and penned chickens, 29,Dec.2016, J-H. Li	C. flaviscutatus	1	0	0	0	1
China, Hainan, Wuzhishan Nature Reserve, LT near gate and penned chickens, 29,Dec.2016, J-H. Li	C. parahumeralis/ C. humeralis	0	0	0	1	1
Adisaptagram, , UV fluorescent tube & LED collection Oct, 2015 India, West Bengal	C. anophelis	783	225	1	5	1014
Adisaptagram, , UV fluorescent tube & LED collection Jun, 2016 India, West Bengal	C. anophelis	166	241	5	1	413
Memari, UV fluorescent tube collection, Aug, Oct 2016,India, West Bengal	C. anophelis	56	5	3	0	64
TOTALS		4158	844	17	131	5304

Table 2 Number of eggs in gravid female specimens from species belonging to Culicoides subg. Trithecoides

Species	Collection details	Eggs
C. flavescens	Thailand, F Pig	40
Trithecoides sp no 1	Solomon Is, Malaita, Kwailatutu, 8°56’24’’S; 160°46’43”E, Green LED trap in cattle paddock, 18.May.2010, L. Halling	38
Trithecoides sp no 1	Solomon Is, Malaita, Kwailatutu, 8°56’24’’S; 160°46’43”E, Green LED trap in cattle paddock, 18.May.2010, L. Halling	88
Trithecoides sp no 1	Solomon Is, Malaita, Kwailatutu, 8°56’24’’S; 160°46’43”E, Green LED trap in cattle paddock, 18.May.2010, L. Halling	28
Trithecoides sp no 1	Solomon Is, Malaita, Kwailatutu, 8°56’24’’S; 160°46’43”E, Green LED trap in cattle paddock, 18.May.2010, L. Halling	56
Trithecoides sp no 1	Solomon Is, Malaita, Kwailatutu, 8°56’24’’S; 160°46’43”E, Green LED trap in cattle paddock, 18.May.2010, L. Halling	44
Trithecoides sp no 1	Solomon Is, Malaita, Kwailatutu, 8°56’24’’S; 160°46’43”E, Green LED trap in cattle paddock, 18.May.2010, L. Halling	56
C. acanthostomus	Malaysia, IMR Grazing grid, Aug 1958, R.Traub	32
C. anophelis	Ceylon, Kalutaluwewa, 19.Feb.1958	66
C. flavescens	Papua New Guinea, Keravat, New Britain, 19.Mar.1958	40
C. paraflavescens	Ceylon, Colombo, Kalutaiuwewa, 19.Feb.1958, (paratype)	64
C. sp near flavescens	Timor-Leste, Ermera, Gleno, .Mar.2017, LT, G.Bellis	114
C. anophelis	Adisaptagram, 22°56’48.08” N, 86°23’10.41”E, UV fluorescent tube & UV LED collection India, West Bengal	156
C. anophelis	Adisaptagram, 22°56’48.08” N, 86°23’10.41”E,UV fluorescent tube & UV LED collection India, West Bengal	148
C. anophelis	Adisaptagram, 22°56’48.08” N, 86°23’10.41”E,UV fluorescent tube & UV LED collection India, West Bengal	73
C. anophelis	Adisaptagram, 22°56’48.08” N, 86°23’10.41”E,UV fluorescent tube & UV LED collection India, West Bengal	82
C. anophelis	Adisaptagram, 22°56’48.08” N, 86°23’10.41”E,UV fluorescent tube & UV LED collection India, West Bengal	59
C. anophelis	Adisaptagram, 22°56’48.08” N, 86°23’10.41”E,UV fluorescent tube & UV LED collection India, West Bengal	154
C. anophelis	Memari, 23°10′43.95″N, 88°05′54.35″E, UV fluorescent tube collection India, West Bengal	178
C. anophelis	Memari, 23°10′43.95″N, 88°05′54.35″E, UV fluorescent tube collection India, West Bengal	124
C. anophelis	Memari, 23°10′43.95″N, 88°05′54.35″E, UV fluorescent tube collection India, West Bengal	90

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Absence of abdominal pigmentation in species of Culicoides Latreille Subgenus Trithecoides Wirth & Hubert: An obstruction to age-grading

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