Characterizing the vector competence of Aphis gossypii, Myzus persicae and Aphis craccivora (Hemiptera: Aphididae) to transmit cotton leafroll dwarf virus to cotton in the United States

Abstract

Cotton leafroll dwarf virus (CLRDV) is a yield-limiting, aphid-transmitted virus that was identified in cotton, Gossypium hirsutum L., in the United States of America in 2017. CLRDV is currently classified in the genus Polerovirus, family Solemoviridae. Although 8 species of aphids (Hemiptera: Aphididae) are reported to infest cotton, Aphis gossypii Glover is the only known vector of CLRDV to this crop. Aphis gossypii transmits CLRDV in a persistent and nonpropagative manner, but acquisition and retention times have only been partially characterized in Brazil. The main objectives of this study were to characterize the acquisition access period, the inoculation access period, and retention times for a U.S. strain of CLRDV and A. gossypii population. A sub-objective was to test the vector competence of Myzus persicae Sulzer and Aphis craccivora Koch. In our study, A. gossypii apterous and alate morphs were able to acquire CLRDV in 30 min and 24 h, inoculate CLRDV in 45 and 15 min, and retain CLRDV for 15 and 23 days, respectively. Neither M. persicae nor A. craccivora acquired or transmitted CLRDV to cotton.

Aphididae, Aphis, virology, plant pathology, plant disease epidemiology, pathogens

Introduction

Cotton, Gossypium hirsutum L., is grown for fiber, feedstock, and oil, and contributes over $7 billion in production alone, in the United States of America (USDA NASS 2021). In 2017, cotton leafroll dwarf virus (CLRDV) (family Solemoviridae and genus Polerovirus) was found in Alabama causing yield loss in cotton and has since been reported in 9 states across the southeastern United States of America (Aboughanem-Sabanadzovic et al. 2019, Avelar et al. 2019, Tabassum et al. 2019, Ali and Mokhtari 2020, Faske et al. 2020, Iriarte et al. 2020, Price et al. 2020, Thiessen et al. 2020, Wang et al. 2020). Three aphid species have been reported as vectors of CLRDV, but only Aphis gossypii Glover, the cotton aphid, is reported to transmit the virus to cotton (Cauquil and Vaissayre 1971, Michelotto and Busoli 2007, Mukherjee et al. 2016, Heilsnis et al. 2022). The transmission parameters have only been partially characterized for A. gossypii on cotton (Michelotto and Busoli 2007). Myzus persicae Sulzer, the green peach aphid, and Aphis craccivora Koch, the cowpea aphid, have been shown to transmit a local strain of CLRDV (Reddy and Kumar 2004) from cotton to chickpea, Cicer arietinum L., in India (Mukherjee et al. 2016). These species have been reported on cotton in the United States of America (Stoetzel et al. 1996) but their vector competence to transmit an American strain of CLRDV to cotton is unknown.

Michelotto and Busoli (2007) showed that both apterous and alate morphs of A. gossypii are capable of transmitting CLRDV-typical, the strain first identified in Argentina and Brazil. Both morphs transmitted the virus in 40 s inoculation access periods (IAPs) and apterae transmitted for up to 12 days after acquisition. The transmission was highest when aphids were allowed 24 h or more acquisition access periods (AAPs) (>45 min) on CLRDV-infected cotton, but different AAPs were not formally evaluated (Michelotto and Busoli 2007). The U.S. population of CLRDV is genetically and phenotypically distinct from South American isolates (Avelar et al. 2020, Ramos-Sobrinho et al. 2021, Tabassum et al. 2021), and are phenotypically different. Strains from the United States of America cause different symptoms and lower levels of yield loss compared to the CLRDV-typical isolate (Brown et al. 2019, Tabassum et al. 2019, Parkash et al. 2021, Mahas et al. 2022), but it is unknown if there are differences in transmissibility between CLRDV strains.

An understanding of the number of vectors, and how quickly viruses can be acquired and inoculated by their vectors, is critical for devising effective strategies to mitigate virus spread. The objectives of this study were to characterize the AAP, IAP, and retention time for alate and apterous morphs of A. gossypii. A secondary objective of this study was to test the vector competence of M. persicae and A. craccivora to acquire and transmit CLRDV to cotton. This is a descriptive biology study to determine whether or not virus acquisition and transmission occurs, and if it does how quickly the virus can be acquired and transmitted, and how long transmission by a viruliferous insect can occur over its adult lifetime.

Materials and Methods

Maintenance of Healthy Cotton Plants

Virus-free cotton plants were grown in a greenhouse in 1.6m × 0.7m × 1.6m cages covered with a 100-micron insect-proof screen (AB Ludvig Svensson, Charlotte, NC) under greenhouse conditions; supplemental lighting was provided in early spring and fall to ensure a minimum 14 h daylength, and temp minimum and maximums did not exceed 18 °C and 43 °C, respectively. Cotton seed variety DP1646 (DeltaPine, Dekalb Genetics Corporation, Dekalb, IL) was sown in 606 cell packs (The HC Companies Inc., Middlefield, OH) filled with peat-lite (PRO-MIX ‘BX’, Quakertown, PA), watered as needed and fertilized wk with 9 g of 20-10-20 fertilizer dissolved in 1.5 l of water (Peters’ Professional 20-10-20 peat-lite special, ICL Group Ltd., Tel Aviv-Yafo, Israel). Seedlings were grown for approximately 3 wk before they were used for either cutting leaf discs used in transmission experiments (described below), or to rear aphid colonies (described below).

Maintenance of CLRDV-Infected Cotton Plants

Cotton plants infected with CLRDV collected from cotton fields in Alabama or Georgia in 2018 were transplanted into 3 gal nursery pots (Nursery Supplies, Chambersburg, PA) and maintained under greenhouse conditions. CLRDV infection was confirmed with RT-PCR (Sharman et al. 2015). Plants were sprayed with an insecticide, M-pede (Gowan, Yuma, AZ), mixed with 75 ml/3.785 l of water to manage occasional insect and mite infestations that occurred so the plants would be arthropod-free when used in experiments. Plants were trimmed to an average height of 81 cm, and bolls, flowers, and excess mainstem branches were removed several times per year to reduce the footprint and height in the greenhouse. Different CLRDV-infected cotton plants were used for each experiment below; CLRDV infection was confirmed 1 to 2 months prior to use in experiments with A. gossypii and A. craccivora.

Rearing A. gossypii and A . craccivora

A nonviruliferous A. gossypii colony was originally collected from a cotton field in Tallassee, AL in 2019. A nonviruliferous A. craccivora colony was originally collected in 2020 from henbit in Auburn, AL. Both species were maintained under greenhouse conditions in BugDorm insect-rearing tents (47.4 cm × 47.5 cm × 93 cm; MegaView Science Co. Ltd., Taiwan). Colonies were sustained on 1–3 true-leaf cotton seedlings. To reduce alatae production, 1 to 2 adult apterae were transferred to new 1–3 true-leaf noninfected cotton seedlings once a week with a fine artist’s paintbrush.

Age-defined cohorts of aphids were generated for transmission experiments by modifying our weekly rearing procedure. For apterae, seedlings newly infested with 2 adults were held for 48 h in BugDorms, after which time the founders were removed to create age-defined cohorts of aphids. For alate aphids, 10 adult apterae were transferred to individual 1–3 true-leaf seedlings to promote crowding conditions and induce alatae production. Seedlings were held for 48 h in BugDorms, after which time the founders were removed to create age-defined cohorts of aphids. Nymphs were then monitored daily for 5–8 days until alatae were observed. Only 1–2-day-old alatae were used for the AAP experiments below, to minimize potential mortality and age-related effects.

A. gossypii Transmission Experiments

A leaf disc assay was used to characterize the transmission parameters according to the methods of Heilsnis et al. (2022). Experimental arenas were constructed from 1.25 oz plastic food cups (Dark, Mason, MI) that were modified to contain insects and leaf discs. Holes (2 cm diameter) were cut from the lids using a soldering iron, and an aphid-proof screen (Equinox, Williamsport, PA) was secured over the hole using hot glue. Plant agar (RPI, Mount Prospect, IL) was mixed with distilled water at 5.5 g/l and heated until dissolved. When the agar was warm to the touch, but had not started to solidify, a 60 ml syringe was used to transfer 5 ml of plant agar to each 1.25 oz cup. Agar was allowed to cool and solidify in the cups and was either used immediately for experiments or covered with cellophane and stored at 5 °C until used.

Expanded, mature leaves were collected from virus-free cotton seedlings with 1–3 true leaves the day of the experiment and cut into 2.5 cm diameter discs using a cork borer, ensuring a vein was included in every disc. Using a 5 ml syringe, a single drop of water was added to each agar cup prior to placing leaf discs abaxial side up on the agar to help hold the leaf disc flat on the agar. This also allowed us to observe feeding aphids. Discs were lightly pressed into the agar to ensure adhesion to the plant agar and prevent curling or desiccation. For all experiments performed below, after the leaf discs were exposed to viruliferous aphids they were held for a total of 5 days in the growth chamber to allow for virus replication before testing for CLRDV using nested RT-PCR according to the methods of Mahas et al. (2022).

Acquisition Access Period Protocol for A. gossypii

To determine the AAP each for adult alatae and apterae, 400 adult aphids were collected from the virus-free colony and starved overnight (>12 h) by holding them in 50 × 9 mm petri dishes with tight-fitting lids (Becton Dickinson, Franklin Lakes, NJ) that were lined with moist filter paper, and modified with aphid-proof screen for ventilation. Aphids were then moved in groups of 80 to each of 5 randomly selected, fully expanded leaves on a CLRDV-infected plant. Aphids were confined on the leaves using (17.78 cm × 15.24 cm × 2.54 cm) bags created from insect-proof mesh (0.78 × 0.25 mm; 50 mesh net anti insect screen, Green-tek, Clinton, WI) that could be sealed around petioles with Velcro.

Aphids were allowed to feed and acquire the virus for the following eight AAPs: 0.5, 1, 2, 4, 6, 12, 24, and 48 h. AAPs occurred at room temp (approximately 23 °C) on an infected plant housed in a dark room for the duration of the experiment to promote aphid settling on the plant. Aphids were confined on leaves using small mesh bags (see description above) for alatae or with 2 weigh boats sealed with parafilm for apterae. Aphids were confined to the leaves during the AAPs to eliminate potential effects of variation in virus accumulation among leaf positions. For alatae, headlamps with red-light were used to observe and collect them in the dark to reduce the likelihood of disturbance and flight due to light. As each AAP ended, the mesh bags were unsealed, 10 aphids were removed from each leaf, and the mesh bags resealed. Groups of 5 aphids were placed on each of the 10 leaf discs; each leaf disc had 1 aphid from each of the 5 CLRDV-infected leaves. Leaf discs with aphids for all experiments described were kept in a growth chamber (Percival Scientific Inc., Perry, IA) at 25 °C, 12:12 light: dark cycle and 50% RH for 5 days before the aphids were manually removed and the leaf discs were frozen in liquid nitrogen and stored in a –80 °C freezer until CLRDV testing was performed. These methods were replicated twice for apterae and 3 times for alatae, due to a lower rate of survival during the experiments. Samples were tested for CLRDV using nested RT-PCR according to the methods of Mahas et al. (2022).

Inoculation Access Period Protocol for A. gossypii

IAP treatment times investigated included 40 s, 15 min, 45 min, 1.5 h, 3 h, 6 h, 12 h, 24 h, and 48 h (Michelotto and Busoli 2007), with an additional ninth IAP of 15 min due to a report that bird cherry-oat aphids, Rhopalosiphum padi Linnaeus, can reach the phloem of black oat seedlings, Avena strigosa Schreb, in 15–30 min (Gray et al. 1991). Aphids were allowed a 4-day AAP on CLRDV-infected plants in Bugdorms in the greenhouse. Then 450 aphids, for each adult alate and apterous morphs, were collected and starved overnight (>12 h) as described above. Groups of 5 aphids were placed on 10 leaf discs per treatment such that each disc had 1 aphid from each of the infested CLRDV-infected leaves used for the AAP to account for potential variation in CLRDV accumulation levels in the host plant. Leaf discs were placed in a growth chamber for the duration of each IAP. At the end of each treatment time, aphids were manually removed with a fine artist’s paintbrush, the leaf discs were returned to the growth chamber for 5 days, and then were frozen in liquid nitrogen and stored in a –80 °C freezer until CLRDV testing. For the IAP treatment of 40 s, aphids were observed under a microscope and timed on a stopwatch for 40 s once a stylet was inserted. These methods were replicated 3 times for alatae and twice for apterae, depending on survival rates.

Retention Time for A. gossypii

Adult viruliferous aphids were collected from CLRDV-infected plants after a 4-day AAP. Virus persistence in the aphid was determined by placing 20 viruliferous apterae and 20 viruliferous alataes individually onto virus-free leaf discs. Every 24 h aphids were transferred to new leaf discs until aphid death. Leaf discs were held for a total of 5 days before freezing at –80 °C and testing for CLRDV using nested RT-PCR according to the methods of Mahas et al. (2022).

Testing Vector Competence of A . craccivora

Vector competence was tested using a 24 h AAP on CLRDV-infected plants in the greenhouse because high mortality of A. craccivora was observed after 24 h. Afterwards, 20 adult aphids were transferred to individual cotton seedlings placed into a 32 oz plastic cup (Fabrikal, Kalamazoo, MI) with another cup inverted as a lid and secured to each other with parafilm. The cup used as a lid had a 2 cm diameter 100-micron screen glued over the bottom to allow for ventilation. These plants were then moved to the growth chamber for a 5-day IAP. Afterwards, aphids were removed from plants and stored at –20 °C until they could be tested for CLRDV acquisition. Seedlings were removed from cups and transferred into Bugdorms in the greenhouse for 7–8 wk until virus testing was performed. To test for virus acquisition by aphids, total RNA was extracted from single aphids using the RNA Tissue MicroPrep kit (Zymo Research, United States of America) according to the manufacturer’s instructions. A total of 40 aphids, which had come from 5 different AAPs, were tested for CLRDV using nested RT-PCR according to Mahas et al. (2022).

Testing Vector Competence of M. Persicae

Nonviruliferous M. persicae which originated from the clone ‘OUR’, was obtained from Juan Alvarez at the University of Idaho, Aberdeen Research and Extension Center, Aberdeen, ID in 2010. These were originally collected from potato plants approximately 30 yr ago and maintained on Chinese cabbage, Brassica pekinensis Ruprecht, under greenhouse conditions. Since 2010, this clone has been maintained at the University of Georgia, GA on Chinese cabbage. Virus acquisition and the inoculation of CLRDV by A. gossypii were used as a positive control. Cotton variety PHY 339 WRF was used for the experiments with M. persicae.

The experiment conducted to assess the ability of M. persicae to acquire CLRDV from infected cotton plants was adopted from the experiment conducted by Pandey et al. (2022) to assess the virus acquisition by A. gossypii. Nonviruliferous adult aphids were confined to the abaxial surface of leaves at the uppermost one-third of the CLRDV-infected cotton plants using clip cages. Aphids were allowed a 72 h AAP and then transferred to a CLRDV nonhost, summer squash for an additional 72 h to facilitate gut clearing. Five aphids were pooled together into 1 sample, and total RNA was extracted from 6 samples per replicate using the Qiagen RNA mini Kit (Valencia, CA) as per the manufacturer’s instructions. Detection of CLRDV by RT-PCR was conducted according to the methods of Pandey et al. (2022). The experiment was replicated 3 times for a total of 90 aphids for each M. persicae and A. gossypii.

The M. persicae-mediated CLRDV-inoculation to cotton plants was assessed in the same way as A. gossypii-mediated CLRDV-inoculation was assessed by Pandey et al. (2022). Approximately 100 adults M. persicae were provided with a 72 h AAP on CLRDV-infected cotton plants. Later, these aphids were transferred to the abaxial surface of leaves from test plants using clip cages for a 72 h IAP. A total of 10 two-true-leaf cotton seedlings were used for each aphid species and were placed inside BugDorm insect-rearing tents in the greenhouse. After the IAP, clip cages were removed and imidacloprid (1% Montana 2F, Rotam, Greensboro, NC, United States of America) was applied to kill any remaining aphids. The topmost young leaves with petioles were collected from each inoculated test plant at 3 wk post-inoculation and tested for CLRDV using RT-PCR as by Pandey et al. (2022). The experiment was conducted 3 times for each aphid species for a total of 30 plants tested for each M. persicae and A. gossypii.

Results

Acquisition Access Period for A. gossypii

A total of 381 leaf discs were tested for the presence of CLRDV: 155 for apterae and 226 for alatae (Table 1). Apterae were able to acquire CLRDV in the 30 min AAP treatment. Alatae did not acquire the virus at any time point less than 24 h (Table 1). For both alate and apterous morphs, an acquisition that resulted in successful transmission was highest at the longest feeding periods of 24 and 48 h, respectively.

Table 1.

Acquisition access periods and inoculation access periods were assessed in apterous and alate morphs of A. gossypii to determine feeding times required to acquire and inoculate cotton leafroll dwarf virus

Acquisition access periods				Inoculation access periods
Duration	Apterae	Alatae	Totals	Duration	Apterae	Alatae	Totals
30 min	1/20a	0/30	1/50	40 s	0/29	0/30	0/59
1 h	2/20	0/30	2/50	15 min	0/19	1/30	1/49
2 h	1/20	0/30	1/50	45 min	1/19	2/30	3/49
4 h	0/20	0/30	0/50	1.5 h	1/19	1/30	2/49
6 h	0/20	0/30	0/50	3 h	2/19	4/30	6/49
12 h	1/20	0/30	1/50	6 h	2/19	6/30	8/49
24 h	1/20	10/30	11/50	12 h	1/19	4/30	5/49
48 h	5/15	1/16	6/31	24 h	4/19	6/30	10/49
–	–	–	–	48 h	6/19	11/30	17/49

^aNumber of leaf discs infected/total tested; each leaf disc was infested with 5 putatively viruliferous aphids.

Inoculation Access Period for A. gossypii

A total of 451 leaf discs were tested for the presence of CLRDV: 181 for apterae and 270 for alatae (Table 1). Apterae inoculated CLRDV within 45 min of feeding time (Table 1), and the highest rate of infection occurred in the IAPs between 3 and 48 h. Alatae aphids inoculated leaf discs in 15 min. Transmission efficiency generally increased with longer IAPs for both morphs tested.

CLRDV Retention for A. gossypii

During the course of 27 days, each alate was determined to transmit at least 1 time (Table 2). Most transmissions occurred between days 5 and 8. Two out of 11 individual alataes were able to transmit up to 23 days after IAP and were observed to transmit on more days during their lifetime than the other alatae (Table 2, #1 and 9). CLRDV transmission was detected for 7 days out of a total of 27 days of life. Others transmitted on days 2 through 4, however, transmission did not always occur on consecutive days. The longest time between transmission events by the same aphid was 9 days (Table 2, #5). Most transmission events detected were intermittent across the aphid’s lifespan.

Table 2.

The duration of cotton leafroll dwarf virus transmission by viruliferous alate A. gossypii individuals during successive 24 h inoculation access periods

Alate	Day
individual	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24	25	26	27	Totala
1	−b	+	−	−	−	+	−	+	−	−	+	−	+	−	+	−	−	−	−	−	−	−	+	−	−	−	−	7/27
2	−	−	−	−	−	−	+	+	−	−	−	−	−															2/13
3	−	−	+	−	+	−	−	−																				2/8
4	−	−	−	−	−	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−								1/20
5	−	−	+	−	+	−	−	−	−	−	−	−	−	−	+	−	−	−	−	−	−	−						3/22
6	−	−	−	−	−	+	−	−	−	+	−																	2/11
7	−	−	−	−	−	+	+	−	−	−	−																	2/11
8	−	−	−	−	+	−	−	+	−	−	−	−	−	−														2/14
9	−	−	−	−	−	+	−	+	−	−	+	−	−	−	−	−	+	+	−	−	−	+	+	−	−	−	−	7/27
10	−	−	−	+	+	−	−	−	+	−	−	−	−	−	−	−	−											3/17
11	−	−	−	−	+	+	+	−	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−				4/24
Totalc	0	1	2	1	5	6	3	4	2	1	2	0	1	0	2	0	1	1	0	0	0	1	2	0	0	0	0

^aTotal number of days an aphid-transmitted CLRDV−AL over the course of its lifespan.

^bResult of transmission in each 24 h feeding period, either positive(+) or negative(−) for CLRDV. Blank cells indicate no result due to aphid death.

^cTotal number of individuals alive and transmitting CLRDV.

Of the 20 apterae used in the study, 14 were transmitted on at least 1 day (Table 3). The highest no. of transmission events observed for an individual was 8 detected between days 1 and 15 (Table 3, #3). The longest time between transmission events by the same aphid was 5 days (Table 3, #3). Apterae had the highest transmission in their first 10 days with over 50% of aphids transmitting on days 1, 2, and 5. The majority of transmission events were intermittent across the aphid’s lifespan.

Table 3.

The duration of cotton leafroll dwarf virus transmission by viruliferous adult apterous A. gossypii individuals during successive 24 h inoculation access periods

Apterous	Day
Individual	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24	25	26	27	Totala
1	−b	−	−	−	+	+	−	−	−	−																		2/10
2	+	+		+	+	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−						5/22
3	+	+	+	+	+	−	−	+	+	−	−	−	−	−	+	−	−	−	−	−	−	−	−	−	−	−		8/26
4	−	+	+	−	−	+	+	−	−	+	−	−	−	−	−	−	−	−	−									5/19
5	+	+	+	+	+	−	−	−	−	−	−																	5/11
6	−	+	+	+	−	+	−	+	−	+	−	−	−	−														6/14
7	+	+	+	−	−	−	+	−	−	−	−	−	−	−	−													4/15
8	−	+	−	−	−	−	+	−	−	−	−	−																2/12
9	−	+	−	+	+	+	−	+	+	−	−	−																6/12
10	+	+	−	−	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	3/27
11	+	−	−	+	−	−	−	−	−	−	−	−	−	−	−													2/15
12	+	+	−	−	+	−	−	−	−	−	−	−	−	−	−	−	−											3/17
13	−	−	−	−	−	−	−	−	−	+	−	−	−	−	−	−	−	−	−									1/19
14	−	−	−	−	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−							1/21
Totalc	7	10	5	6	8	5	3	3	2	3	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0	0

^aTotal number of days an aphid-transmitted CLRDV−AL over the course of its lifespan

^bResult of transmission in each 24 h feeding period, either positive (+) or negative (−) for CLRDV. Blank cells indicate no result due to aphid death.

^cTotal number of individuals alive and transmitting CLRDV.

Vector Competence of A . craccivora

A total of 46 plants were tested for the presence of CLRDV 7–8 wk after the IAP. None of the plants tested positive for CLRDV. In addition, CLRDV acquisition was not detected in any of the 40 A. craccivora aphids tested. These results indicate that A. craccivora did not acquire CLRDV from the virus-infected cotton plants.

Vector Competence of M . persicae

Thirty cotton plants were infested with potentially viruliferous A. gossypii and M. persicae. CLRDV infection was detected in 28/30 (93.33%) cotton plants inoculated using A. gossypii, but no CLRDV was detected in plants (0/30) inoculated using M. persicae. Of 18 pools of aphids collected, 16 (88.88%) of A. gossypii samples tested positive for CLRDV, but no samples of M. persicae tested positive for CLRDV. These results indicate that M. persicae did not acquire CLRDV from infected cotton plants.

Discussion

The results of this study provide new information about the transmission parameters of CLRDV by A. gossypii and show that M. persicae and A. craccivora did not acquire or transmit CLRDV from cotton. CLRDV transmission by a U.S. population of A. gossypii corroborates previously published partial IAP and retention time’s characterization from Brazil (Michelotto and Busoli 2007). Overall, apterae acquired CLRDV more quickly than alatae (Table 1, AAP), whereas alatae inoculated more quickly in the IAP (Table 1, IAP), and more frequently in both the IAP and CLRDV retention experiments (Tables 1 and 2). New data on AAPs with apterae showed a minimum acquisition time of 30 min by adult apterae, and 24 h by alatae, which are required for successful transmission to occur. Longer AAPs were shown to increase the transmission efficiency, with the highest occurring at 48 and 24 h for apterae and alatae, respectively. Once an aphid acquired the virus, both apterae and alatae were capable of inoculating for 15 and 23 days, respectively, which is longer than has been reported in Brazil. This suggests that aphids can transmit the virus most of their adult lives, which may have important implications for primary and secondary spread events, especially for alatae, as they can be carried by wind during long-distance dispersal (CABI 2021).

The large difference between the time required for A. gossypii apterae (30 min) and alatae (24 h) to acquire CLRDV may be explained by feeding and dispersal behavior differences described for apterous and alate morphs (Boquel et al. 2011). Apterae are the primary colonizers and are more likely to engage in prolonged, undisturbed feeding periods (Williams and Dixon 2007, Boquel et al. 2011). Alatae is produced as a result of declining host quality and overcrowding and tend toward dispersal to seek out new hosts. Alatae in this study did not always readily settle onto infected plants to feed and would orient to light sources if not held in a dark room. It has also been reported that some aphid species require a flight period before they will settle and feed (Johnson 1958). CLRDV is reported to be phloem-limited (Silva et al. 2008), and electrical penetration graph studies show that it can take aphids 30 min or longer of feeding to reach the phloem of host plants (Prado and Tjallingii 1997, Gonzalez-Mas et al. 2019). Although placing the alatae in darkness encouraged them to settle, they may not have engaged in long-duration phloem feeding events required to acquire CLRDV (Jiménez et al. 2020). The same behaviors of alatae could explain their decreased survival in the 48 h treatments. From an epidemiological perspective, these results suggest that reproductive hosts of A. gossypii that are also hosts of CLRDV may play a larger role in virus spread because all life stages could acquire CLRDV before alatae disperse, whereas alatae that developed on a noninfected host would have to settle and feed on a CLRDV-infected host for an extended period of time to become viruliferous.

During the retention time experiment, it was observed that viruliferous A. gossypii do not transmit continuously that is, during each successive 24 h period of feeding (Tables 2 and 3). The variation in time between detection of successful transmission by the same aphid can be explained by the persistent, circulative nature of this virus. Persistent viruses transmitted by aphids may have a latent period lasting anywhere from minutes to hours, depending on the species and aphids often transmit for their entire lifespan (Hogenhout et al. 2008), even if they do not support virus replication (Gray et al. 2014). Persistently transmitted virions travel passively in the hemocoel from the gut to reach the accessory salivary gland (ASG), and therefore, reach the ASG at different times (Katis et al. 2007), before they can be egested into plant tissue during feeding. Therefore, multiple inoculation events from the same aphid are possible.

Though M. persicae and A. craccivora have been shown to transmit CLRDV to chickpeas from cotton in India (Mukherjee et al. 2016), no acquisition of CLRDV from cotton was observed, and therefore, neither transmitted CLRDV to cotton seedlings. Both species are reported to feed on cotton in the United States of America (Stoetzel et al. 1996), and A. craccivora can occasionally be found on seedling cotton in Alabama (personal observation). In these experiments, A. craccivora survival dropped sharply after 24 h on mature cotton plants infected with CLRDV. This may be due to an age-related effect of cotton because although the colony was reared on cotton seedlings, mortality was observed if the colony was reared for consecutive weeks on the same plants, and mortality increased once the cotton reached the 5–6 true-leaf stage (personal observation). Terpenoids such as gossypol have been shown to increase in cotton over time, especially as a result of wounds, fungi, and insects (Hunter et al. 1978, Opitz et al. 2008, Scheffler 2016), and have been shown to decrease both the lifespan and fecundity of adult aphids (Du et al. 2004). This study does not rule out vector competence if cotton was a poor acquisition host for these species, but future experiments would be needed to test acquisition from alternate hosts of CLRDV and each aphid species.

It is important to understand the acquisition, inoculation, and retention times for viruses, and any differences that may occur between alate and apterous morphs. The information reported here on 3 of the 8 aphid species reported on cotton in the United States of America (Stoetzel et al. 1996) can also be used as methods to test additional aphid species for vector competence. Dispersals of alatae aphids to fields are responsible for the primary spread into crops, whereas both morphs may contribute to secondary spread within a field (Williams and Dixon 2007). Information about transmission parameters will be valuable for designing management strategies to reduce the spread of CLRDV. The quick transmission times observed may explain why insecticides used to kill aphids do not reduce the final incidence of CLRDV in cotton (Mahas et al. 2022). Future studies are needed to better understand aphid–plant–virus interactions, including testing vector competence of additional aphid species and the contribution of primary versus secondary spread to final virus incidence in cotton.

Supplementary material

Supplementary material is available at Journal of Economic Entomology online .

Acknowledgments

The authors would like to thank Adam Kesheimer and the research assistants in the Jacobson and Srinivasan labs for their assistance with greenhouse plant maintenance and the preparation of supplies used in these experiments.

Funding

Funding was provided by Cotton Incorporated 19-212, The Foundation for Food and Agricultural Research ROAR-0000000020, Agricultural Research Service, U.S. Department of Agriculture Agreement No. 58-6010-0-011, Hatch Project AL-1021180, and GA GEO-0671.

Notes on Contributors

Brianna Heilsnis (Conceptualization-Equal, Data curation-Equal, Formal analysis-Equal, Investigation-Equal, Methodology-Equal, Writing – original draft-Supporting, Writing – review & editing-Equal), Jessica Mahas (Data curation-Equal, Formal analysis-Equal, Investigation-Equal, Methodology-Equal, Writing – original draft-Equal, Writing – review & editing-Equal), Kassie Conner (Formal analysis-Equal, Investigation-Equal, Methodology-Equal, Resources-Equal, Writing – review & editing-Supporting), Sudeep Pandey (Conceptualization-Equal, Data curation-Equal, Formal analysis-Equal, Investigation-Equal, Methodology-Equal, Writing – original draft-Supporting, Writing – review & editing-Supporting), Wilson Clark (Investigation-Supporting, Writing – review & editing-Supporting), Jenny Koebernick (Conceptualization-Equal, Funding acquisition-Equal, Methodology-Equal, Project administration-Equal, Writing – review & editing-Supporting), Rajagopalbabu Srinivasan (Conceptualization-Equal, Funding acquisition-Equal, Methodology-Equal, Project administration-Equal, Supervision-Supporting, Writing – review & editing-Supporting), Kathleen Martin (Methodology-Equal, Resources-Equal, Writing – review & editing-Supporting), Alana Jacobson (Conceptualization-Equal, Funding acquisition-Equal, Methodology-Equal, Project administration-Lead, Resources-Equal, Supervision-Equal, Validation-Equal, Writing – review & editing-Equal)

Author notes

Co-first authors contributed equally to completing experiments and manuscript preparation.